Therapeutic inhibitor of vascular smooth muscle cells

ABSTRACT

Methods are provided for inhibiting or treating stenosis or restenosis following vascular trauma or disease in a mammalian host, comprising administering to the host a therapeutically effective amount of a therapeutic agent via a catheter. Also provided is a catheter adapted for administering a therapeutically effective amount of a therapeutic agent to a mammalian host for inhibiting or treating stenosis or restenosis.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.09/910,388, filed Jul. 20, 2001, which is a continuation of U.S. patentapplication Ser. No. 09/470,662, filed Dec. 22, 1999, now U.S. Pat. No.6,268,390, which is a continuation of U.S. patent application Ser. No.09/113,733, filed Jul. 10, 1998, now U.S. Pat. No. 6,074,659, which is acontinuation of U.S. patent application Ser. No. 08/450,793, filed May25, 1995, now U.S. Pat. No. 5,811,447, which is a continuation of U.S.patent application Ser. No. 08/062,451, filed May 13, 1993, abandoned,which is a continuation-in-part of U.S. patent application Ser. No.08/011,669, filed Jan. 28, 1993, abandoned, filed the entire disclosureof each of which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention relates generally to therapeutic methods involvingsurgical or intravenous introduction of binding partners directed tocertain target cell populations, such as smooth muscle cells, cancercells, somatic cells requiring modulation to ameliorate a disease stateand effector cells of the immune system, particularly for treatingconditions such as stenosis following vascular trauma or disease,cancer, diseases resulting from hyperactivity or hyperplasia of somaticcells and diseases that are mediated by immune system effector cells.Surgical or intravenous introduction of active agents capable ofaltering the proliferation or migration of smooth muscle cells orcontraction of smooth muscle proteins is also described. The inventionalso relates to the direct or targeted delivery of therapeutic agents tovascular smooth muscle cells that results in dilation and fixation ofthe vascular lumen (biological stenting effect). Combined administrationof a cytocidal conjugate and a sustained release dosage form of avascular smooth muscle cell inhibitor is also disclosed. Mechanisms forin vivo vascular smooth muscle cell proliferation modulation, agentsthat impact those mechanisms and protocols for the use of those agentsare discussed.

BACKGROUND OF THE INVENTION

Percutaneous transluminal coronary angioplasty (PTCA) is widely used asthe primary treatment modality in many patients with coronary arterydisease. PTCA can relieve myocardial ischemia in patients with coronaryartery disease by reducing lumen obstruction and improving coronaryflow. The use of this surgical procedure has grown rapidly, with 39,000procedures performed in 1983, nearly 150,000 in 1987, 200,000 in 1988,250,000 in 1989, and over 500,000 PTCAs per year are estimated by 1994(1, 2, 3). Stenosis following PTCA remains a significant problem, withfrom 25% to 35% of the patients developing restenosis within 1 to 3months. Restenosis results in significant morbidity and mortality andfrequently necessitates further interventions such as repeat angioplastyor coronary bypass surgery. No surgical intervention or post-surgicaltreatment (to date) has proven effective in preventing restenosis.

The processes responsible for stenosis after PTCA are not completelyunderstood but may result from a complex interplay among severaldifferent biologic agents and pathways. Viewed in histological sections,restenotic lesions may have an overgrowth of smooth muscle cells in theintimal layers of the vessel (3). Several possible mechanisms for smoothmuscle cell proliferation after PTCA have been suggested (1, 2, 4, 5).

Compounds that reportedly suppress smooth muscle proliferation in vitro(4, 6, 7) may have undesirable pharmacological side effects when used invivo. Heparin is an example of one such compound, which reportedlyinhibits smooth muscle cell proliferation in vitro but when used in vivohas the potential adverse side effect of inhibiting coagulation. Heparinpeptides, while having reduced anti-coagulant activity, have theundesirable pharmacological property of having a short pharmacologicalhalf-life. Attempts have been made to solve such problems by using adouble balloon catheter, i.e., for regional delivery of the therapeuticagent at the angioplasty site (e.g., 8; U.S. Pat. No. 4,824,436), and byusing biodegradable materials impregnated with a drug, i.e., tocompensate for problems of short half-life (e.g., 9; U.S. Pat. No.4,929,602).

Verrucarins and Roridins are trichothecene drugs produced as secondarymetabolites by the soil fungi Myrothecium verrucaria and Myrotheciumroridium. Verrucarin is a macrocyclic triester. Roridin is a macrocyclicdiester of verrucarol (10). As a group, the trichothecenes arestructurally related to sesquiterpenoid mycotoxins produced by severalspecies of fungi and characterized by the 12,13-epoxytrichothec-9-enebasic structure. Their cytotoxic activity to eukaryotic cells is closelycorrelated with their ability to bind to the cell, to be internalized,and to inhibit protein and macromolecular synthesis in the cell.

At least five considerations would, on their face, appear to precludeuse of inhibitory drugs to prevent stenosis resulting from overgrowth ofsmooth muscle cells. First, inhibitory agents may have systemic toxicitythat could create an unacceptable level of risk for patients withcardiovascular disease. Second, inhibitory agents might interfere withvascular wound healing following surgery and that could either delayhealing or weaken the structure or elasticity of the newly healed vesselwall. Third, inhibitory agents killing smooth muscle cells could damagesurrounding endothelium and/or other medial smooth muscle cells. Deadand dying cells also release mitogenic agents that might stimulateadditional smooth muscle cell proliferation and exacerbate stenosis.Fourth, delivery of therapeutically effective levels of an inhibitoryagent may be problematic from several standpoints: namely, a) deliveryof a large number of molecules into the intercellular spaces betweensmooth muscle cells may be necessary, i.e., to establish favorableconditions for allowing a therapeutically effective dose of molecules tocross the cell membrane; b) directing an inhibitory drug into the properintracellular compartment, i.e., where its action is exerted, may bedifficult to control; and, c) optimizing the association of theinhibitory drug with its intracellular target, e.g., a ribosome, whileminimizing intercellular redistribution of the drug, e.g. to neighboringcells, may be difficult. Fifth, because smooth muscle cell proliferationtakes place over several weeks it would appear a priori that theinhibitory drugs should also be administered over several weeks, perhapscontinuously, to produce a beneficial effect.

As is apparent from the foregoing, many problems remain to be solved inthe use of inhibitory drugs, including cytotoxic agents, to effectivelytreat smooth muscle cell proliferation. It would be highly advantageousto develop new methods for inhibiting stenosis due to proliferation ofvascular smooth muscle cells following traumatic injury to vessels suchas occurs during vascular surgery. In addition, delivery of compoundsthat produce inhibitory effects of extended duration to the vascularsmooth muscle cells would be advantageous. Local administration of suchsustained release compounds would also be useful in the treatment ofother conditions where the target cell population is accessible by suchadministration.

SUMMARY OF THE INVENTION

In one aspect of the invention, new therapeutic methods and therapeuticconjugates are provided for inhibiting vascular smooth muscle cells in amammalian host. The therapeutic conjugates contain a vascular smoothmuscle binding protein or peptide that binds in a specific manner to thecell membranes of a vascular smooth muscle cell or an interstitialmatrix binding protein/peptide that binds in a specific manner tointerstitial matrix (e.g., collagen) of the artery wall, coupled to atherapeutic agent that inhibits the activity of the cell. In oneembodiment, inhibition of cellular activity results in reducing,delaying, or eliminating stenosis after angioplasty or other vascularsurgical procedures. The therapeutic conjugates of the invention achievethese advantageous effects by associating with vascular smooth musclecells and pericytes, which may transform into smooth muscle cells. Thetherapeutic conjugate may contain: (1) therapeutic agents that altercellular metabolism or are inhibitors of protein synthesis, cellularproliferation, or cell migration; (2) microtubule and microfilamentinhibitors that affect morphology or increases in cell volume; and/or(3) inhibitors of extracellular matrix synthesis or secretion. In onerepresentative embodiment, the conjugates include a cytotoxictherapeutic agent that is a sesquiterpenoid mycotoxin such as averrucarin or a roridin. Other embodiments involve cytostatictherapeutic agents that inhibit DNA synthesis and proliferation at dosesthat have a minimal effect on protein synthesis such as protein kinaseinhibitors (e.g., staurosporin), suramin, transforming growthfactor-beta (TGF-beta) activators or production stimulators such astrans-2-[4-(1,2-diphenyl-1-butenyl)phenoxy]-N,N-dimethylethylamine(tamoxifen), TGF-beta itself, and nitric oxide releasing compounds(e.g., nitroglycerin) or analogs or functional equivalents thereof.Other moieties that inhibit cell division and are, therefore, useful inthe practice of the present invention, include, for example, taxol andanalogs thereof such as taxotere. In addition, therapeutic agents thatinhibit the contraction or migration of smooth muscle cells and maintainan enlarged luminal area following, for example, angioplasty trauma(e.g., the cytochalasins, such as cytochalasin B, cytochalasin C,cytochalasin D, taxol or analogs thereof such as taxotere or the like)are also contemplated for use in accordance with the present invention.Other aspects of the invention relate to vascular smooth muscle bindingproteins that specifically associate with a chondroitin sulfateproteoglycan (CSPG) expressed on the membranes of a vascular smoothmuscle cell, and in a preferred embodiment this CSPG has a molecularweight of about 250 kDaltons. In preferred embodiments the vascularsmooth muscle binding protein binds to a CSPG target on the cell surfacewith an association constant of at least 10⁻⁴ M. In another preferredembodiment, the vascular smooth muscle binding protein contains asequence of amino acids found in the Fab, Fv or CDR (complementaritydetermining regions) of monoclonal antibody NR-AN-01 or functionalequivalents thereof.

Other aspects of the invention include methods for inhibiting stenosis,e.g., following angioplasty in a mammalian host, by administering to ahuman or animal subject in need of such treatment a therapeuticallyeffective dosage of a therapeutic conjugate of the invention. In onerepresentative embodiment, the dosage of therapeutic conjugate may beadministered with an infusion catheter, to achieve a 10⁻³ M to 10⁻¹² Mconcentration of said therapeutic conjugate at the site ofadministration in a blood vessel.

The present invention also contemplates therapeutic methods andtherapeutic dosage forms involving sustained release of therapeuticagent to target cells. Preferably, the target cells are vascular smoothmuscle cells, cancer cells, somatic cells requiring modulation toameliorate a disease state and cells involved in immune system-mediateddiseases that are accessible by local administration of the dosage form.Consequently, the methods and dosage forms of this aspect of the presentinvention are useful for inhibiting vascular smooth muscle cells in amammalian host, employing a therapeutic agent that inhibits the activityof the cell (e.g., proliferation, contraction, migration or the like)but does not kill the cell and, optionally, a vascular smooth musclecell binding protein. Also, the methods and dosage forms of this aspectof the present invention are useful for inhibiting target cellproliferation or killing such target cells, employing a therapeuticagent that inhibits proliferation or is cytotoxic to the target cellsand, optionally, a target cell binding protein. In addition, the methodsand dosage forms of this aspect of the present invention are useful fordelivering cytostatic, cytocidal or metabolism modulating therapeuticagents to target cells, such as effector cells of the immune system,that are accessible by local administration of the dosage form,optionally employing a target cell binding protein. Finally, dosageforms of the present invention are useful to reduce or eliminatepathological proliferation or hyperactivity of normal tissue (i.e.,somatic cells).

The dosage forms of the present invention are preferably eithernon-degradable microparticulates or nanoparticulates or biodegradablemicroparticulates or nanoparticulates. More preferably, themicroparticles or nanoparticles are formed of a polymer containingmatrix that biodegrades by random, nonenzymatic, hydrolytic scissioning.A particularly preferred structure is formed of a mixture ofthermoplastic polyesters (e.g., polylactide or polyglycolide) or acopolymer of lactide and glycolide components. The lactide/glycolidestructure has the added advantage that biodegradation thereof formslactic acid and glycolic acid, both normal metabolic products ofmammals.

Preferable therapeutic agents dispersed within the microparticulates ornanoparticulates are those exhibiting inhibition of a therapeuticallysignificant target cell activity without killing the target cell, ortarget cell killing activity. For treatment of restenosis of vascularsmooth muscle cells, useful therapeutic agents inhibit target cellactivity (e.g., proliferation or migration) without killing the targetcells. Preferred therapeutic moieties for this purpose are proteinkinase inhibitors (e.g., staurosporin or the like), TGF-beta productionor activation stimulators, such as tamoxifen or TGF-beta itself, taxolor analogs thereof (e.g., taxotere), smooth muscle migration and/orcontraction inhibitors (e.g., the cytochalasins, such as cytochalasin B,cytochalasin C, cytochalasin D or the like), suramin, and nitricoxide-releasing compounds, such as nitroglycerin, or analogs orfunctional equivalents thereof. In cancer therapy, useful therapeuticagents inhibit proliferation or are cytotoxic to the target cells.Preferred therapeutic moieties for this purpose are TGF-beta productionor activation stimulators, such as tamoxifen or TGF-beta itself, taxolor analogs thereof (e.g., taxotere), Roridin A and Pseudomonas exotoxin,or analogs or functional equivalents thereof. For treatment of immunesystem-modulated diseases, such as arthritis, useful therapeutic agentsdeliver cytostatic, cytocidal or metabolism-modulating therapeuticagents to target cells that are accessible by local administration ofthe dosage form. Preferred therapeutic moieties for this purpose areRoridin A, Pseudomonas exotoxin, suramin, TGF-beta production oractivation stimulators, such as tamoxifen or TGF-beta itself, taxol oranalogs thereof (e.g., taxotere) and protein kinase inhibitors (e.g.,staurosporin), sphingosine, or analogs or functional equivalentsthereof. For treatment of pathologically proliferating normal tissues(e.g., proliferative vitreoretinopathy, corneal pannus and the like),anti-proliferative agents or antimigration agents are preferred (e.g.,cytochalasins, taxol or analogs thereof, somatostatin, somatostatinanalogs, N-ethylmaleimide, antisense oligonucleotides, TGF-betaproduction or activation stimulators, such as tamoxifen or TGF-betaitself and the like).

The dosage forms of the present invention are optionally targeted to arelevant target cell population by a binding protein or peptide.Preferred binding proteins/peptides of the present invention arevascular smooth muscle cell binding protein, tumor cell binding proteinand immune system effector cell binding protein. Preferred vascularsmooth muscle cell binding proteins specifically associate with achondroitin sulfate proteoglycan (CSPG) expressed on the membranes of avascular smooth muscle cell, and in a preferred embodiment this CSPG hasa molecular weight of about 250 kDaltons. In preferred embodiments, thevascular smooth muscle binding protein binds to a CSPG target on thecell surface with an association constant of at least 10⁻⁴ M. In otherpreferred embodiments, the vascular smooth muscle binding proteincontains a sequence of amino acids found in the Fab, Fv or CDR(complementarity determining regions) of monoclonal antibody NR-AN-01 orfunctional equivalents thereof. Other preferred binding peptides usefulin this embodiment of the present invention include those that localizeto intercellular stroma and matrix located between and among vascularsmooth muscle cells. Preferred binding peptides of this type arespecifically associated with collagen, reticulum fibers or otherintercellular matrix compounds. Preferred tumor cell binding proteinsare associated with surface cell markers expressed by the target tumorcell population or cytoplasmic epitopes thereof. Preferred immunesystem-modulated target cell binding proteins are associated with cellsurface markers of the target immune system effector cells orcytoplasmic epitopes thereof. Binding peptides/proteins of the presentinvention also target pathologically proliferating normal tissues.

The present invention also provides therapeutic methods and therapeuticdosage forms involving administration of free (i.e., non-targeted ornon-binding partner associated) therapeutic agent to target cells.Preferably, the target cells are vascular smooth muscle cells and thetherapeutic agent is an inhibitor of vascular smooth muscle cellcontraction, allowing the normal hydrostatic pressure to dilate thevascular lumen. Such contraction inhibition may be achieved by actininhibition, which is preferably achievable and sustainable at a lowerdose level than that necessary to inhibit protein synthesis.Consequently, the vascular smooth muscle cells synthesize proteinrequired to repair minor cell trauma and secrete interstitial matrix,thereby facilitating the fixation of the vascular lumen in a dilatedstate near its maximal systolic diameter. This phenomenon constitutes abiological stenting effect that diminishes or prevents the undesirablerecoil mechanism that occurs in up to 25% of the angioplasty proceduresclassified as successful based on an initial post-procedural angiogram.Cytochalasins (which inhibit the polymerization of G- to F-actin which,in turn, inhibits the migration and contraction of vascular smoothmuscle cells) are the preferred therapeutic agents for use in thisembodiment of the present invention. Free therapeutic agent protocols ofthis type effect a reduction, a delay, or an elimination of stenosisafter angioplasty or other vascular surgical procedures. Preferably,free therapeutic agent is administered directly or substantiallydirectly to vascular smooth muscle tissue. Such administration ispreferably effected by an infusion catheter, to achieve a 10⁻³ M to10⁻¹² M concentration of said therapeutic agent at the site ofadministration in a blood vessel.

Another embodiment of the present invention incorporates administrationof a cytocidal targeted conjugate to destroy proliferating vascularsmooth muscle cells involved in vascular stenosis. The mitogenic agentsreleased after this biological arteromyectomy are prevented fromstimulating the remaining viable vascular smooth muscle cells toproliferate and restenose the vessel by administration of theanti-contraction, anti-migration or anti-proliferative sustained releaseagents of the present invention.

TGF-beta, TGF-beta activator and TGF-beta production stimulatorsustained release dosage forms of the present invention may be employedin the prevention or treatment of conditions characterized byinappropriate proliferation of smooth muscle cells, such as theprevention or reduction of restenosis following angioplasty or othervascular trauma. TGF-beta or such TGF-beta activators and productionstimulators inhibit abnormal proliferation of smooth muscle cells. Apreferred TGF-beta activator/production stimulator is trans2-[4-(1,2-diphenyl-1-butenyl) phenoxy-N,N-dimethylethylamine.

The amount of TGF-beta, TGF-beta activator or TGF-beta productionstimulator therapeutic or prophylactic agent administered in sustainedrelease dosage forms is selected to treat vascular trauma of differingseverity, with smaller doses being sufficient to treat lesser vasculartrauma such as in the prevention of vascular rejection following graftor transplant. Such dosage forms are also amenable to chronic use forprophylactic purposes with respect to disease states involvingproliferation of vascular smooth muscle cells over time (e.g.,atherosclerosis, coronary heart disease, thrombosis, myocardialinfarction, stroke, smooth muscle neoplasms such as leiomyoma andleiomyosarcoma of the bowel and uterus, uterine fibroid or fibroma andthe like). For the prevention/treatment of restenosis, for example, alarge dose (optionally, in sustained release form) is administeredbefore or during an angioplasty procedure, followed by a sustainedrelease dosage form designed to release smaller, follow up doses overtime to maintain an anti-proliferative effect for a time sufficient tosubstantially reduce the risk of or prevent restenosis. A preferredtherapeutic protocol duration for this purpose is from about 3 to about26 weeks.

Further provided is a method for upregulating cellular mRNA coding forTGF-beta. Cells (e.g., smooth muscle cells) amenable to such metabolicmanipulation are identified in the manner described herein and areexposed to sustained release formulation of an effective amount of aTGF-beta mRNA regulator (i.e., a subset of TGF-beta productionstimulators). In this manner, TGF-beta production is stimulated, therebyinhibiting the abnormal proliferation of smooth muscle cells.

Free TGF-beta, TGF-beta production stimulator or TGF-beta activator maybe employed in combination protocols to prevent or combat conditionscharacterized by abnormal proliferation of smooth muscle cells. In onesuch protocol, systemic TGF-beta or TGF-beta activator or TGF-betaproduction stimulator is administered prior to a local (e.g., viacatheter) administration of a cytotoxic agent (e.g., free cytotoxicagent, a cytotoxic agent-containing conjugate, or a cytotoxicagent-containing sustained release dosage form). The TGF-beta, TGF-betaactivator or TGF-beta production stimulator decreases the effect of theproliferative stimulus provided upon cell death caused by the action ofthe cytotoxic agent. In this manner, proliferating smooth muscle cellscan be killed without causing rampant proliferation of the remainingcells. Preferably, systemic TGF-beta or TGF-beta activator or TGF-betaproduction stimulator administrations occur following cytotoxic agentadministration to maintain an anti-proliferative environment. Also,localized TGF-beta, TGF-beta activator or TGF-beta production stimulatoradministration can optionally be carried out in conjunction with thelocalized delivery of cytotoxic agent. Similarly, TGF-beta, TGF-betaactivator or TGF-beta production stimulator may be administered incombination with one or more cytostatic agents.

DESCRIPTION OF THE DRAWINGS

FIG. 1A is a photomicrograph of vascular smooth muscle cells of a24-year old male patient.

FIG. 1B is a photomicrograph of vascular smooth muscle cells in anartery of a 24-year-old male patient with vascular smooth muscle bindingprotein bound to the cell surface and membrane. The patient received thevascular smooth muscle binding protein by i.v. administration 4 daysbefore the arterial tissue was prepared for histology.

FIG. 2 depicts a first scheme for chemical coupling of a therapeuticagent to a vascular smooth muscle binding protein.

FIG. 3 depicts a second scheme for chemical coupling of a therapeuticagent to a vascular smooth muscle binding protein.

FIG. 4A graphically depicts experimental data showing rapid binding ofvascular smooth muscle binding protein to marker-positive test cells invitro.

FIG. 4B graphically depicts experimental data showing rapid binding ofvascular smooth muscle binding protein to vascular smooth muscle cellsin vitro.

FIG. 5A presents graphically experimental data showing undesirablecytotoxicity of even low levels of therapeutic conjugate (i.e.,RA-NR-AN-01), and the free RA therapeutic agent, when vascular smoothmuscle cells were treated for 24 hours in vitro.

FIG. 5B graphically presents experimental data showing the effects ofRA-NR-AN-01 therapeutic conjugate on metabolic activity ofmarker-positive and -negative cells. The data show undesirablenonspecific cytotoxicity of the conjugate for all these cells in a 24hour treatment in vitro. The non-specificity results from extracellularhydrolysis of the coupling ligand which exposes the tested cells to freedrug.

FIG. 6A graphically depicts experimental data showing undesirablenonspecific cytotoxicity of PE-NR-AN-01 therapeutic conjugate formarker-positive and marker-negative test cells after 24 hours oftreatment in vitro, even though the 24 hour treatment was followed by anovernight recovery period prior to testing the metabolic activity.

FIG. 6B depicts experimental data showing nonspecific cytotoxicity ofthe free PE therapeutic agent on marker-positive and -negative testcells after 24 hours of treatment in vitro.

FIG. 7A graphically presents experimental data showing that a short 5minute “pulse” treatment, i.e., instead of 24 hours, followed byexposure to [³H]leucine, with free RA therapeutic agent beingnonspecifically cytotoxic, i.e., for control HT29 marker-negative cells,but, in contrast, the RA-NR-AN-01 therapeutic conjugate is not cytotoxicin this “pulse” treatment.

FIG. 7B presents graphically experimental data showing that free RAtherapeutic agent is nonspecifically cytotoxic for control HT29marker-negative cells, even in a 5′ “pulse” treatment followed by a 24hour recovery period prior to [³H]leucine exposure, but, in contrast,the RA-NR-AN-01 therapeutic conjugate is not cytotoxic to cells.

FIG. 7C presents graphically results of experiments showing that “pulse”treatment of cells in vitro with the RA-NR-AN-01 therapeutic conjugateinhibits cellular activity in marker-positive A375 cells, as measured byprotein synthesis.

FIG. 7D presents graphically experimental data showing that “pulse”treatment of cells in vitro with the RA-NR-AN-01 therapeutic conjugatedid not exert long-lasting inhibitory effects on cellular activity inmarker-positive cells, since protein synthesis in A375 cells was notinhibited when the cells were allowed an overnight recovery period priorto testing in vitro.

FIG. 8A presents graphically experimental data showing that while a“pulse” treatment of cells in vitro with free RA therapeutic agent wasnon-specifically cytotoxic, the RA-NR-AN-01 therapeutic conjugate didnot exert long-lasting inhibitory effects on cellular activity invascular smooth muscle cells, as evidenced by metabolic activity in BO54cells that were allowed a 48 hour recovery period prior to testing.

FIG. 8B graphically depicts experimental data similar to those presentedin FIG. 8A, above, but using a second marker-positive cell type, namelyA375, the data show that “pulse” treatment with the RA-NR-AN-01therapeutic conjugate did not exert long-lasting inhibitory effects oncellular activity, as measured by metabolic activity in A375 cells thatwere allowed a 48 hour recovery period prior to testing.

FIG. 8C graphically depicts results similar to those presented in FIG.8A and FIG. 8B, above, but using a marker-negative control cell type,namely HT29. The results show that the “pulse” treatment with theRA-NR-AN-01 therapeutic conjugate did not exert long-lasting inhibitoryeffects on the cellular activity of marker-negative control cells, asmeasured by metabolic activity in HT29 cells that were allowed a 48 hourrecovery period prior to testing.

FIG. 9A shows stenosis due to intimal smooth muscle cell proliferationin a histological section of an untreated artery 5 weeks afterangioplasty in an animal model.

FIG. 9B shows inhibition of stenosis in a histological section of anartery treated with therapeutic conjugate at 5 weeks after angioplastyin an animal model.

FIG. 10A graphically depicts experimental data comparing proteinsynthesis and DNA synthesis inhibition capability of suramin withrespect to vascular smooth muscle cells.

FIG. 10B graphically depicts experimental data comparing proteinsynthesis and DNA synthesis inhibition capability of staurosporin withrespect to vascular smooth muscle cells.

FIG. 10C graphically depicts experimental data comparing proteinsynthesis and DNA synthesis inhibition capability of nitroglycerin withrespect to vascular smooth muscle cells.

FIG. 10D graphically depicts experimental date comparing proteinsynthesis and DNA synthesis inhibition capability of cytochalasin B withrespect to vascular smooth muscle cells.

FIG. 11 shows a tangential section parallel to the inner surface of asmooth muscle cell which is magnified 62,500 times and is characterizedby numerous endocytic vesicles, several of which contain antibody coatedgold beads in the process of being internalized by the cell in vitro.

FIG. 12 shows a smooth muscle cell which is magnified 62,500 times andis characterized by a marked accumulation of gold beads in lysosomes at24 hours following exposure of the cell to the beads in vitro.

FIG. 13 shows a smooth muscle cell which is magnified 62,500 times andis characterized by accumulation of gold beads in lysosomes in vivo.

FIG. 14 depicts an in vivo dose response study of the effect ofcytochalasin B on the luminal area of pig femoral arteries.

FIGS. 15 and 16 depict pathways for the modulation of vascular smoothmuscle cell proliferation in vivo.

DETAILED DESCRIPTION OF THE INVENTION

As used herein the following terms have the meanings as set forth below:

“Therapeutic conjugate” means a vascular smooth muscle or aninterstitial matrix binding protein coupled (e.g., optionally through alinker) to a therapeutic agent.

“Therapeutic agent” includes any moiety capable of exerting atherapeutic or prophylactic effect in the practice of the presentinvention.

“Target” and “marker” are used interchangeably in describing theconjugate aspects of the present invention to mean a molecule recognizedin a specific manner by the matrix or vascular smooth muscle bindingprotein, e.g., an antigen, polypeptide antigen or cell surfacecarbohydrate (e.g., a glycolipid, glycoprotein, or proteoglycan) that isexpressed on the cell surface membranes of a vascular smooth muscle cellor a matrix structure.

“Epitope” is used to refer to a specific site within the “target”molecule that is bound by the matrix or smooth muscle binding protein,e.g., a sequence of three or more amino acids or saccharides.

“Coupled” is used to mean covalent or non-covalent chemical association(i.e., hydrophobic as through van der Waals forces or charge-chargeinteractions) of the matrix or vascular smooth muscle binding proteinwith the therapeutic agent. Due to the nature of the therapeutic agentsemployed, the binding proteins will normally be associated with thetherapeutic agents by means of covalent bonding.

“Linker” means an agent that couples the matrix or smooth muscle bindingprotein to a therapeutic agent, e.g., an organic chemical coupler.

“Migration” of smooth muscle cells means movement of these cells in vivofrom the medial layers of a vessel into the intima, such as may also bestudied in vitro by following the motion of a cell from one location toanother (e.g., using time-lapse cinematography or a video recorder andmanual counting of smooth muscle cell migration out of a defined area inthe tissue culture over time).

“Proliferation,” i.e., of smooth muscle cells or cancer cells, meansincrease in cell number, i.e., by mitosis of the cells.

“Abnormal or Pathological or Inappropriate Proliferation” meansdivision, growth or migration of cells occurring more rapidly or to asignificantly greater extent than typically occurs in a normallyfunctioning cell of the same type.

“Expressed” means mRNA transcription and translation with resultantsynthesis, glycosylation, and/or secretion of a polypeptide by a cell,e.g., CSPG synthesized by a vascular smooth muscle cell or pericyte.

“Macrocyclic trichothecene” is intended to mean any one of the group ofstructurally related sesquiterpenoid macrocyclic mycotoxins produced byseveral species of fungi and characterized by the12,13-epoxytrichothec-9-ene basic structure, e.g., verrucarins androridins that are the products of secondary metabolism in the soil fungiMyrothecium verrucaria and Myrothecium roridium.

“Sustained release” means a dosage form designed to release atherapeutic agent therefrom for a time period ranging from about 3 toabout 21 days. Release over a longer time period is also contemplated asa “sustained release” dosage form of the present invention.

“Dosage form” means a free (non-targeted or non-binding partnerassociated) therapeutic agent formulation, as well as sustained releasetherapeutic formulations, such as those incorporating microparticulateor nanoparticulate, biodegradable or non-biodegradable polymericmaterial capable of binding to one or more binding proteins or peptidesto deliver a therapeutic moiety dispersed therein to a target cellpopulation.

“Staurosporin” includes staurosporin, a protein kinase C inhibitor ofthe following formula,

as well as diindoloalkaloids having one of the following generalstructures:

More specifically, the term “staurosporin” includes K-252 (see, forexample, Japanese Patent Application No. 62,164,626), BMY-41950 (U.S.Pat. No. 5,015,578), UCN-01 (U.S. Pat. No. 4,935,415), TAN-999 (JapanesePatent Application No. 01,149,791), TAN-1030A (Japanese PatentApplication No. 01,246,288), RK-286C (Japanese Patent Application No.02,258,724) and functional equivalents and derivatives thereof.Derivatives of staurosporin include those discussed in Japanese PatentApplication Nos. 03,72,485; 01,143,877; 02,09,819 and 03,220,194, aswell as in PCT International Application Nos. WO 89 07,105 and WO 9109,034 and European Patent Application Nos. EP 410,389 and EP 296,110.Derivatives of K-252, a natural product, are known. See, for example,Japanese Patent Application Nos. 63,295,988; 62,240,689; 61,268,687;62,155,284; 62,155,285; 62,120,388 and 63,295,589, as well as PCTInternational Application No. WO 88 07,045 and European PatentApplication No. EP 323,171.

“Cytochalasin” includes fungal metabolites exhibiting an inhibitoryeffect on target cellular metabolism, including prevention ofcontraction or migration of vascular smooth muscle cells. Preferably,cytochalasins inhibit the polymerization of monomeric actin (G-actin) topolymeric form (F-actin), thereby inhibiting cell functions requiringcytoplasmic microfilaments. Cytochalasins typically are derived fromphenylalanine (cytochalasins), tryptophan (chaetoglobosins), or leucine(aspochalasins), resulting in a benzyl, indol-3-yl methyl or isobutylgroup, respectively, at position C-3 of a substitutedperhydroisoindole-1-one moiety (Formula V or VI).

The perhydroisoindole moiety in turn contains an 11-, 13- or 14-atomcarbocyclic- or oxygen-containing ring linked to positions C-8 and C-9.All naturally occurring cytochalasins contain a methyl group at C-5; amethyl or methylene group at C-12; and a methyl group at C-14 or C-16.Exemplary molecules include cytochalasin A, cytochalasin B, cytochalasinC, cytochalasin D, cytochalasin E, cytochalasin F, cytochalasin G,cytochalasin H, cytochalasin J, cytochalasin K, cytochalasin L,cytochalasin M, cytochalasin N, cytochalasin O, cytochalasin P,cytochalasin Q, cytochalasin R, cytochalasin S, chaetoglobosin A,chaetoglobosin B, chaetoglobosin C, chaetoglobosin D, chaetoglobosin E,chaetoglobosin F, chaetoglobosin G, chaetoglobosin J, chaetoglobosin K,deoxaphomin, proxiphomin, protophomin, zygosporin D, zygosporin E,zygosporin F, zygosporin G, aspochalasin B, aspochalasin C, aspochalasinD and the like, as well as functional equivalents and derivativesthereof. Certain cytochalasin derivatives are set forth in JapanesePatent Nos. 72 01,925; 72 14,219; 72 08,533; 72 23,394; 72 01924; and 7204,164. Cytochalasin B is used in this description as a prototypicalcytochalasin.

As referred to herein, “tamoxifen” includestrans-2-[4-(1,2-diphenyl-1-butenyl)phenoxy]-N,N-dimethyl-ethylaminewhich is capable of enhancing the production or activation of TGF-beta.The activated form of TGF-beta, in turn, inhibits vascular smooth musclecell proliferation. Evidence exists that tamoxifen also acts tostabilize or organize areas of smooth muscle cell trauma. Thisorganization/stabilization may stem from a blockage of smooth musclecell maturation. Functional equivalents and derivatives of theaforementioned chemical compound are also included within the scope ofthe term “tamoxifen” for the purposes of this disclosure. Exemplarytamoxifen functional equivalents are plasmin, heparin, compounds capableof reducing the level or inactivating the lipoprotein Lp(a) or theglycoprotein apolipoprotein(a) and derivatives or analogs thereof.

As referred to herein, “TGF-beta” includes transforming growthfactor-beta as well as functional equivalents, derivatives and analogsthereof. TGF-beta is a polypeptide produced in a latent propeptide formhaving, at this time, no identified biological activity. To be renderedactive and, therefore, capable of inhibiting vascular smooth muscle cellproliferation, the propeptide form of TGF-beta must be cleaved.Functional equivalents of TGF-beta are, for example, moieties capable ofdisrupting cyclin-dependent protein kinase (CDK) transformation from aslow migrating form to a rapid migrating form, disrupting CDK-cyclincomplex formation or activation or the like.

“TGF-beta activator” includes moieties capable of directly or indirectlyactivating the latent form of TGF-beta to the active form thereof.Plasmin, plasmin activators, tamoxifen as well as analogs, derivativesor functional equivalents thereof are exemplary TGF-beta activatorsuseful in the practice of the present invention.

“TGF-beta production stimulator” includes moieties capable of directlyor indirectly stimulating the production of TGF-beta (generally thelatent form thereof). Such TGF-beta production stimulators may beTGF-beta mRNA regulators (i.e., moieties that increase the production ofTGF-beta mRNA), enhancers of TGF-beta mRNA expression or the like.

“Direct” action implies that a first moiety acts on a second moiety,e.g., a TGF-beta activator acts on the latent form of TGF-beta. Suchdirect action of TGF-beta production stimulators indicates that cellsupon which the production stimulate acts to increase TGF-beta mRNAproduction or expression of TGF-beta.

“Indirect” action implies that a first moiety acts on one or moreintermediate moieties, one of which ultimately acts on the secondmoiety, e.g., a TGF-beta activator acts on a moiety that itself orthrough one or more other moieties acts on latent TGF-beta. Suchindirect action of TGF-beta production stimulators indicates that thestimulators act on a moiety that itself or through one or more othermoieties acts on a population of cells to stimulate the production ofTGF-beta mRNA or the expression of TGF-beta.

As referred to herein, “taxol” includes taxol, analogs thereof such astaxotere as well as functional equivalents or derivatives thereof. Taxolis readily taken up into cells and stabilizes such cells against celldivision.

As referred to herein, a “cytostatic agent” includes moieties capable ofinhibiting one or more pathological activities of target cells for atime sufficient to achieve a therapeutic benefit.

As referred to herein, smooth muscle cells and pericytes include thosecells derived from the medial layers of vessels and adventitia vesselswhich proliferate in intimal hyperplastic vascular sites followinginjury, such as that caused during PTCA.

Characteristics of smooth muscle cells include a histological morphology(under light microscopic examination) of a spindle shape with an oblongnucleus located centrally in the cell with nucleoli present andmyofibrils in the sarcoplasm. Under electron microscopic examination,smooth muscle cells have long slender mitochondria in the juxtanuclearsarcoplasm, a few tubular elements of granular endoplasmic reticulum,and numerous clusters of free ribosomes. A small Golgi complex may alsobe located near one pole of the nucleus. The majority of the sarcoplasmis occupied by thin, parallel myofilaments that may be, for the mostpart, oriented to the long axis of the muscle cell. These actincontaining myofibrils may be arranged in bundles with mitochondriainterspersed among them. Scattered through the contractile substance ofthe cell may also be oval dense areas, with similar dense areasdistributed at intervals along the inner aspects of the plasmalemma.

Characteristics of pericytes include a histological morphology (underlight microscopic examination) characterized by an irregular cell shape.Pericytes are found within the basement membrane that surrounds vascularendothelial cells and their identity may be confirmed by positiveimmuno-staining with antibodies specific for alpha smooth muscle actin(e.g., anti-alpha-sm1, Biomakor, Rehovot, Israel), HMW-MAA, and pericyteganglioside antigens such as MAb 3G5 (11); and, negative immuno-stainingwith antibodies to cytokeratins (i.e., epithelial and fibroblastmarkers) and von Willdebrand factor (i.e., an endothelial marker). Bothvascular smooth muscle cells and pericytes are positive byimmunostaining with the NR-AN-01 monoclonal antibody.

The therapeutic conjugates and dosage forms of the invention are usefulfor inhibiting the activity of vascular smooth muscle cells, e.g., forreducing, delaying, or eliminating stenosis following angioplasty. Asused herein the term “reducing” means decreasing the intimal thickeningthat results from stimulation of smooth muscle cell proliferationfollowing angioplasty, either in an animal model or in man. “Delaying”means delaying the time until onset of visible intimal hyperplasia(e.g., observed histologically or by angiographic examination) followingangioplasty and may also be accompanied by “reduced” restenosis.“Eliminating” restenosis following angioplasty means completely“reducing” and/or completely “delaying” intimal hyperplasia in a patientto an extent which makes it no longer necessary to surgically intervene,i.e., to re-establish a suitable blood flow through the vessel by repeatangioplasty, atheroectomy, or coronary artery bypass surgery. Theeffects of reducing, delaying, or eliminating stenosis may be determinedby methods routine to those skilled in the art including, but notlimited to, angiography, ultrasonic evaluation, fluoroscopic imaging,fiber optic endoscopic examination or biopsy and histology. Thetherapeutic conjugates of the invention achieve these advantageouseffects by specifically binding to the cellular membranes of smoothmuscle cells and pericytes.

Therapeutic conjugates of the invention are obtained by coupling avascular smooth muscle binding protein to a therapeutic agent. In thetherapeutic conjugate, the vascular smooth muscle binding proteinperforms the function of targeting the therapeutic conjugate to vascularsmooth muscle cells or pericytes, and the therapeutic agent performs thefunction of inhibiting the cellular activity of the smooth muscle cellor pericyte.

Therapeutic dosage forms (sustained release-type) of the presentinvention exhibit the capability to deliver therapeutic agent to targetcells over a sustained period of time. Therapeutic dosage forms of thisaspect of the present invention may be of any configuration suitable forthis purpose. Preferred sustained release therapeutic dosage formsexhibit one or more of the following characteristics:

-   -   microparticulate (e.g., from about 0.5 micrometers to about 100        micrometers in diameter, with from about 0.5 to about 2        micrometers more preferred) or nanoparticulate (e.g., from about        1.0 nanometer to about 1000 nanometers in diameter, with from        about 50 to about 250 nanometers more preferred), free flowing        powder structure;    -   biodegradable structure designed to biodegrade over a period of        time between from about 3 to about 180 days, with from about 10        to about 21 days more preferred, or non-biodegradable structure        to allow therapeutic agent diffusion to occur over a time period        of between from about 3 to about 180 days, with from about 10 to        about 21 days preferred;    -   biocompatible with target tissue and the local physiological        environment into which the dosage form is being administered,        including biocompatible biodegradation products;    -   facilitate a stable and reproducible dispersion of therapeutic        agent therein, preferably to form a therapeutic agent-polymer        matrix, with active therapeutic agent release occurring through        one or both of the following routes: (1) diffusion of the        therapeutic agent through the dosage form (when the therapeutic        agent is soluble in the polymer or polymer mixture forming the        dosage form); or (2) release of the therapeutic agent as the        dosage form biodegrades; and    -   capability to bind with one or more cellular and/or interstitial        matrix epitopes, with from about 1 to about 10,000 binding        protein/peptide-dosage form bonds preferred and with a maximum        of about 1 binding peptide-dosage form per 150 square angstroms        of particle surface area more preferred. The total number bound        depends upon the particle size used. The binding proteins or        peptides are capable of coupling to the particulate therapeutic        dosage form through covalent ligand sandwich or non-covalent        modalities as set forth herein.

Nanoparticulate sustained release therapeutic dosage forms of preferredembodiments of the present invention are biodegradable and bind to thevascular smooth muscle cells and enter such cells primarily byendocytosis. The biodegradation of such nanoparticulates occurs overtime (e.g., 10 to 21 days) in prelysosomic vesicles and lysosomes. Thepreferred larger microparticulate therapeutic dosage forms of thepresent invention bind to the target cell surface or interstitialmatrix, depending on the binding protein or peptide selected, andrelease the therapeutic agents for subsequent target cell uptake withonly a few of the smaller microparticles entering the cell byphagocytosis. A practitioner in the art will appreciate that the precisemechanism by which a target cell assimilates and metabolizes a dosageform of the present invention depends on the morphology, physiology andmetabolic processes of those cells.

The size of the targeted sustained release therapeutic particulatedosage forms is also important with respect to the mode of cellularassimilation. For example, the smaller nanoparticles can flow with theinterstitial fluid between cells and penetrate the infused tissue untilit binds to the normal or neoplastic tissue that the bindingprotein/peptide is selected to target. This feature is important, forexample, because the nanoparticles follow lymphatic drainage channelsfrom infused primary neoplastic foci, targeting metastatic foci alongthe lymphatic tract. The larger microparticles tend to be more easilytrapped interstitially in the infused primary tissue.

Preferable sustained release dosage forms of the present invention arebiodegradable microparticulates or nanoparticulates. More preferably,biodegradable microparticles or nanoparticles are formed of a polymercontaining matrix that biodegrades by random, nonenzymatic, hydrolyticscissioning to release therapeutic agent, thereby forming pores withinthe particulate structure.

Polymers derived from the condensation of alpha hydroxycarboxylic acidsand related lactones are preferred for use in the present invention. Aparticularly preferred moiety is formed of a mixture of thermoplasticpolyesters (e.g., polylactide or polyglycolide) or a copolymer oflactide and glycolide components, such as poly(lactide-co-glycolide). Anexemplary structure, a random poly(DL-lactide-co-glycolide), is shownbelow, with the values of x and y being manipulable by a practitioner inthe art to achieve desirable microparticulate or nanoparticulateproperties.

Other agents suitable for forming particulate dosage forms of thepresent invention include polyorthoesters and polyacetals (PolymerLetters, 18:293, 1980) and polyorthocarbonates (U.S. Pat. No. 4,093,709)and the like.

Preferred lactic acid/glycolic acid polymer containing matrixparticulates of the present invention are prepared by emulsion-basedprocesses, that constitute modified solvent extraction processes such asthose described by Cowsar et al., “Poly(Lactide-Co-Glycolide)Microcapsules for Controlled Release of Steroids,” Methods Enzymology,112:101-116, 1985 (steroid entrapment in microparticulates); Eldridge etal., “Biodegradable and Biocompatible Poly(DL-Lactide-Co-Glycolide)Microspheres as an Adjuvant for Staphylococcal Enterotoxin B ToxoidWhich Enhances the Level of Toxin-Neutralizing Antibodies,” Infectionand Immunity, 5:2978-2986, 1991 (toxoid entrapment); Cohen et al.,“Controlled Delivery Systems for Proteins Based on Poly(Lactic/GlycolicAcid) Microspheres,” Pharmaceutical Research, 8(6):713-720, 1991 (enzymeentrapment); and Sanders et al., “Controlled Release of a LuteinizingHormone-Releasing Hormone Analogue from Poly(D,L-Lactide-Co-Glycolide)Microspheres,” J. Pharmaceutical Science, 73(9):1294-1297, 1984 (peptideentrapment).

In general, the procedure for forming particulate dosage forms of thepresent invention involves dissolving the polymer in a halogenatedhydrocarbon solvent, dispersing a therapeutic agent solution (preferablyaqueous) therein, and adding an additional agent that acts as a solventfor the halogenated hydrocarbon solvent but not for the polymer. Thepolymer precipitates out from the polymer-halogenated hydrocarbonsolution onto droplets of the therapeutic agent containing solution andentraps the therapeutic agent. Preferably the therapeutic agent issubstantially uniformly dispersed within the sustained release dosageform of the present invention. Following particulate formation, they arewashed and hardened with an organic solvent. Water washing and aqueousnon-ionic surfactant washing steps follow, prior to drying at roomtemperature under vacuum.

For biocompatibility purposes, particulate dosage forms, characterizedby a therapeutic agent dispersed therein in matrix form, are sterilizedprior to packaging, storage or administration. Sterilization may beconducted in any convenient manner therefor. For example, theparticulates can be irradiated with gamma radiation, provided thatexposure to such radiation does not adversely impact the structure orfunction of the therapeutic agent dispersed in the therapeuticagent-polymer matrix or the binding protein/peptide attached thereto. Ifthe therapeutic agent or binding protein/peptide is so adverselyimpacted, the particulate dosage forms can be produced under sterileconditions.

Release of the therapeutic agent from the particulate dosage forms ofthe present invention can occur as a result of both diffusion andparticulate matrix erosion. Biodegradation rate directly impactstherapeutic agent release kinetics. The biodegradation rate is regulableby alteration of the composition or structure of the sustained releasedosage form. For example, alteration of the lactide/glycolide ratio inpreferred dosage forms of the present invention can be conducted, asdescribed by Tice et al., “Biodegradable Controlled-Release ParenteralSystems,” Pharmaceutical Technology, pp. 26-35, 1984; by inclusion ofpolymer hydrolysis modifying agents, such as citric acid and sodiumcarbonate, as described by Kent et al., “Microencapsulation of WaterSoluble Active Polypeptides,” U.S. Pat. No. 4,675,189; by altering theloading of therapeutic agent in the lactide/glycolide polymer, thedegradation rate being inversely proportional to the amount oftherapeutic agent contained therein, and by judicious selection of anappropriate analog of a common family of therapeutic agents that exhibitdifferent potencies so as to alter said core loadings; and by variationof particulate size, as described by Beck et al.,“Poly(DL-Lactide-Co-Glycolide)/Norethisterone Microcapsules: AnInjectable Biodegradable Contraceptive,” Biol. Reprod., 28:186-195,1983, or the like. All of the aforementioned methods of regulatingbiodegradation rate influence the intrinsic viscosity of the polymercontaining matrix, thereby altering the hydration rate thereof.

The preferred lactide/glycolide structure is biocompatible with themammalian physiological environment. Also, these preferred sustainedrelease_dosage forms have the advantage that biodegradation thereofforms lactic acid and glycolic acid, both normal metabolic products ofmammals.

Functional groups required for binding protein/peptide-particulatedosage form bonding to the particles, are optionally included in theparticulate structure, along with the non-degradable or biodegradablepolymeric units. Functional groups that are exploitable for this purposeinclude those that are reactive with peptides, such as carboxyl groups,amine groups, sulfhydryl groups and the like. Preferred bindingenhancement moieties include the terminal carboxyl groups of thepreferred (lactide-glycolide) polymer containing matrix or the like.

Useful vascular smooth muscle binding protein is a polypeptide,peptidic, or mimetic compound (as described below) that is capable ofbinding to a target or marker on a surface component of an intact ordisrupted vascular smooth muscle cell in such a manner that allows foreither release of therapeutic agent extracellularly in the immediateinterstitial matrix with subsequent diffusion of therapeutic agent intothe remaining intact smooth muscle cells and/or internalization by thecell into an intracellular compartment of the entire targetedbiodegradable moiety, permitting delivery of the therapeutic agent.Representative examples of useful vascular smooth muscle bindingproteins include antibodies (e.g., monoclonal and polyclonalaffinity-purified antibodies, F(ab′)₂, Fab′, Fab, and Fv fragmentsand/or complementarity determining regions (CDR) of antibodies orfunctional equivalents thereof, (e.g., binding peptides and the like));growth factors, cytokines, and polypeptide hormones and the like; andmacromolecules recognizing extracellular matrix receptors (e.g.,integrin and fibronectin receptors and the like).

Other preferred binding peptides useful in targeting the dosage formembodiment of the present invention include those that localize tointercellular stroma and matrix located between and among vascularsmooth muscle cells. Such binding peptides deliver the therapeutic agentto the interstitial space between the target cells. The therapeuticagent is released into such interstitial spaces for subsequent uptake bythe vascular smooth muscle cells. Preferred binding peptides of thistype are associated with epitopes on collagen, extracellularglycoproteins such as tenascin, reticulum and elastic fibers and otherintercellular matrix material.

Preferred tumor cell binding peptides are associated with epitopes ofmyc, ras, bcr/Abl, erbB and like gene products, as well as mucins,cytokine receptors such as IL-6, EGF, TGF and the like, which bindingpeptides localize to certain lymphomas (myc), carcinomas such as coloncancer (ras), carcinoma (erbB), adenocarcinomas (mucins), breast cancerand hepatoma (IL-6 receptor), and breast cancer (EGF and TGF),respectively. Preferred immune system effector cell-binding peptides areanti-TAC, IL-2 and the like, which localize to activated T cells andmacrophages, respectively. Other preferred binding proteins/peptidesuseful in the practice of the present invention include moieties capableof localizing to pathologically proliferating normal tissues, such aspericytes of the intraocular vasculature implicated in degenerative eyedisease.

Therapeutic agents of the invention are selected to inhibit a cellularactivity of a vascular smooth muscle cell, e.g., proliferation,migration, increase in cell volume, increase in extracellular matrixsynthesis (e.g., collagens, proteoglycans, and the like), or secretionof extracellular matrix materials by the cell. Preferably, thetherapeutic agent acts either: a) as a “cytostatic agent” to prevent ordelay cell division in proliferating cells by inhibiting replication ofDNA (e.g., a drug such as adriamycin, staurosporin, tamoxifen or thelike), or by inhibiting spindle fiber formation (e.g., a drug such ascolchicine) and the like; or b) as an inhibitor of migration of vascularsmooth muscle cells from the medial wall into the intima, e.g., an“anti-migratory agent” such as a cytochalasin; or c) as an inhibitor ofthe intracellular increase in cell volume (i.e., the tissue volumeoccupied by a cell; a “cytoskeletal inhibitor” or “metabolicinhibitor”); or d) as an inhibitor that blocks cellular proteinsynthesis and/or secretion or organization of extracellular matrix(i.e., an “anti-matrix agent” such as tamoxifen).

Representative examples of “cytostatic agents” include, e.g., modifiedtoxins, methotrexate, adriamycin, radionuclides (e.g., such as disclosedin Fritzberg et al., U.S. Pat. No. 4,897,255), protein kinase inhibitors(e.g., staurosporin), stimulators of the production or activation ofTGF-beta, including tamoxifen and functional equivalents or derivativesthereof, TGF-beta or functional equivalents, derivatives or analogsthereof, taxol or analogs thereof (e.g., taxotere), inhibitors ofspecific enzymes (such as the nuclear enzyme DNA topoisomerase II andDNA polymerase, RNA polymerase, adenyl guanyl cyclase), superoxidedismutase inhibitors, terminal deoxynucleotidyl-transferase, reversetranscriptase, antisense oligonucleotides that suppress smooth musclecell proliferation and the like, which when delivered into a cellularcompartment at an appropriate dosage will act to impair proliferation ofa smooth muscle cell or pericyte without killing the cell. Otherexamples of “cytostatic agents” include peptidic or mimetic inhibitors(i.e., antagonists, agonists, or competitive or non-competitiveinhibitors) of cellular factors that may (e.g., in the presence ofextracellular matrix) trigger proliferation of smooth muscle cells orpericytes: e.g., cytokines (e.g., interleukins such as IL-1), growthfactors, (e.g., PDGF, TGF-alpha or -beta, tumor necrosis factor, smoothmuscle- and endothelial-derived growth factors, i.e., endothelin, FGF),homing receptors (e.g., for platelets or leukocytes), and extracellularmatrix receptors (e.g., integrins). Representative examples of usefultherapeutic agents in this category of cytostatic agents for smoothmuscle proliferation include: subfragments of heparin,triazolopyrimidine (Trapidil; a PDGF antagonist), lovastatin, andprostaglandins E1 or 12.

Representative examples of “anti-migratory agents” include inhibitors(i.e., agonists and antagonists, and competitive or non-competitiveinhibitors) of chemotactic factors and their receptors (e.g., complementchemotaxins such as C5a, C5a desarg or C4a; extracellular matrixfactors, e.g., collagen degradation fragments), or of intracellularcytoskeletal proteins involved in locomotion (e.g., actin, cytoskeletalelements, and phosphatases and kinases involved in locomotion).Representative examples of useful therapeutic agents in this category ofanti-migratory agents include: caffeic acid derivatives and nilvadipine(a calcium antagonist), and steroid hormones. Preferred anti-migratorytherapeutic agents are the cytochalasins.

Representative examples of “cytoskeletal inhibitors” include colchicine,vinblastin, cytochalasins, taxol and the like that act on microtubuleand microfilament networks within a cell.

Representative examples of “metabolic inhibitors” include staurosporin,trichothecenes, and modified diphtheria and ricin toxins, Pseudomonasexotoxin and the like. In a preferred embodiment, the therapeuticconjugate is constructed with a therapeutic agent that is a simpletrichothecene or a macrocyclic trichothecene, e.g., a verrucarin orroridin. Trichothecenes are drugs produced by soil fungi of the classFungi imperfecti or isolated from Baccharus megapotamica (Bamburg, J. R.Proc. Molec. Subcell. Biol. 8:41-110, 1983; Jarvis & Mazzola, Acc. Chem.Res. 15:338-395, 1982). They appear to be the most toxic molecules thatcontain only carbon, hydrogen and oxygen (Tamm, C. Fortschr. Chem. Org.Naturst. 31:61-117, 1974). They are all reported to act at the level ofthe ribosome as inhibitors of protein synthesis at the initiation,elongation, or termination phases.

There are two broad classes of trichothecenes: those that have only acentral sesquiterpenoid structure and those that have an additionalmacrocyclic ring (simple and macrocyclic trichothecenes, respectively).The simple trichothecenes may be subdivided into three groups (i.e.,Group A, B, and C) as described in U.S. Pat. Nos. 4,744,981 and4,906,452 (incorporated herein by reference). Representative examples ofGroup A simple trichothecenes include: Scirpene, Roridin C,dihydrotrichothecene, Scirpen-4,8-diol, Verrucarol, Scirpentriol, T-2tetraol, pentahydroxyscirpene, 4-deacetylneosolaniol, trichodermin,deacetylcalonectrin, calonectrin, diacetylverrucarol,4-monoacetoxyscirpenol, 4,15-diacetoxyscirpenol,7-hydroxydiacetoxyscirpe nol, 8-hydroxydiacetoxy-scirpenol(Neosolaniol), 7,8-dihydroxydiacetoxyscirpenol7-hydroxy-8-acetyldiacetoxyscirpenol, 8-acetylneosolaniol, NT-1, NT-2,HT-2, T-2, and acetyl T-2 toxin.

Representative examples of Group B simple trichothecenes include:Trichothecolone, Trichothecin, deoxynivalenol, 3-acetyldeoxynivalenol,5-acetyldeoxynivalenol, 3,15-diacetyldeoxynivalenol, Nivalenol,4-acetylnivalenol (Fusarenon-X), 4,15-idacetylnivalenol,4,7,15-triacetylnivalenol, and tetra-acetylnivalenol. Representativeexamples of Group C simple trichothecenes include: Crotocol andCrotocin. Representative macrocyclic trichothecenes include VerrucarinA, Verrucarin B, Verrucarin J (Satratoxin C), Roridin A, Roridin D,Roridin E (Satratoxin D), Roridin H, Satratoxin F, Satratoxin G,Satratoxin H, Vertisporin, Mytoxin A, Mytoxin C, Mytoxin B, Myrotoxin A,Myrotoxin B, Myrotoxin C, Myrotoxin D, Roritoxin A, Roritoxin B, andRoritoxin D. In addition, the general “trichothecene” sesquiterpenoidring structure is also present in compounds termed “baccharins” isolatedfrom the higher plant Baccharis megapotamica, and these are described inthe literature, for instance as disclosed by Jarvis et al. (Chemistry ofAlleopathy, ACS Symposium Series No. 268: ed. A. C. Thompson, 1984, pp.149-159).

Representative examples of “anti-matrix agents” include inhibitors(i.e., agonists and antagonists and competitive and non-competitiveinhibitors) of matrix synthesis, secretion and assembly, organizationalcross-linking (e.g., transglutaminases cross-linking collagen), andmatrix remodeling (e.g., following wound healing). A representativeexample of a useful therapeutic agent in this category of anti-matrixagents is colchicine, an inhibitor of secretion of extracellular matrix.Another example is tamoxifen for which evidence exists regarding itscapability to organize and/or stabilize as well as diminish smoothmuscle cell proliferation following angioplasty. The organization orstabilization may stem from the blockage of vascular smooth muscle cellmaturation in to a pathologically proliferating form.

For the sustained release dosage form embodiments of the presentinvention, therapeutic agents preferably are those that inhibit vascularsmooth muscle cell activity without killing the cells (i.e., cytostatictherapeutic agents). Another way to define a cytostatic agent is amoiety capable of inhibiting one or more pathological activities of thetarget cells for a time sufficient to achieve a therapeutic benefit.Preferred therapeutic agents for this purpose exhibit one or more of thefollowing capabilities: to inhibit DNA synthesis prior to proteinsynthesis inhibition or to inhibit migration of vascular smooth musclecells into the intima. These therapeutic agents do not significantlyinhibit protein synthesis (i.e., do not kill the target cells) and,therefore, facilitate cellular repair and matrix production to stabilizethe vascular wall lesion caused by angioplasty, by reducing smoothmuscle cell proliferation.

Exemplary of such preferred therapeutic agents are protein kinaseinhibitors, such as staurosporin (staurosporine is available from SigmaChemical Co., St. Louis, Mo.) cytochalasins, such as cytochalasin B(Sigma Chemical Co.), and suramin (FBA Pharmaceuticals, West Haven,Conn.), as well as nitroglycerin (DuPont Pharmaceuticals, Inc., Manuti,Puerto Rico) or analogs or functional equivalents thereof. Thesecompounds are cytostatic and have been shown to exert minimal proteinsynthesis inhibition and cytotoxicity at concentrations wheresignificant DNA synthesis inhibition occurs (see Example 8 and FIGS.10A-10D). Other exemplary preferred therapeutic agents are TGF-betaactivators or production stimulators, such as tamoxifen and functionalequivalents or derivatives thereof. TGF-beta and its analogs,derivatives or functional equivalents may also be employed. Taxol andits analogs, derivatives or functional equivalents are also useful inthe practice of the present invention. A useful protocol for identifyingtherapeutic agents useful in sustained release dosage form embodimentsof the present invention is set forth in Example 8, for example. Apractitioner in the art is capable of designing substantially equivalentexperimental protocols for making such an identification for differenttarget cell populations, such as adherent monolayer target cell types.

Other embodiments of the present invention involve agents that arecytotoxic to cancer cells. Preferred agents for these embodiments areRoridin A, Pseudomonas exotoxin and the like or analogs or functionalequivalents thereof. A plethora of such therapeutic agents, includingradioisotopes and the like, have been identified and are known in theart. In addition, protocols for the identification of cytotoxic moietiesare known and employed routinely in the art.

Modulation of immune system-mediated disease effector cells can also beaccomplished using the sustained release dosage forms of the presentinvention. Such modulation is preferably conducted with respect todiseases having an effector cell population that is accessible throughlocal sustained release dosage form administration. Therapeutic moietieshaving the requisite modulating activity, e.g., cytocidal, cytostatic,metabolism modulation or like activity upon lymphorecticular cells inthe treatment of arthritis (intra-articular administration), sprue (oraladministration), uveitis and endophthalmitis (intra-ocularadministration) and keratitis (sub-conjunctival administration), areidentifiable using techniques that are known in the art. These agentscan also be used to reduce hyperactivity of epithelial glands andendocrine organs that results in multiple disorders. Preferred agentsfor these embodiments include Roridin A, Pseudomonas exotoxin, suramin,protein kinase inhibitors (e.g., staurosporin), TGF-beta and TGF-betaactivators or production stimulators such as tamoxifen, taxol and thelike, or analogs or functional equivalents thereof.

Other preferred therapeutic agents useful in the practice of the presentinvention include moieties capable of reducing or eliminatingpathological proliferation, migration or hyperactivity of normaltissues. Exemplary of such therapeutic agents are those capable ofreducing or eliminating hyperactivity of corneal epithelium and stroma,pathological proliferation or prolonged contraction of smooth musclecells or pericytes of the intraocular vasculature implicated indegenerative eye disease resulting from hyperplasia or decreasedvascular lumen area. Preferred agents for this purpose are TGF-beta andTGF-beta activators or production stimulators such as tamoxifen, taxoland analogs thereof, staurosporin and cytochalasin B as well asfunctional equivalents or derivatives thereof.

Vascular smooth muscle binding proteins of the invention bind to targetson the surface of vascular smooth muscle cells. It will be recognizedthat specific targets, e.g., polypeptides or carbohydrates,proteoglycans and the like, that are associated with the cell membranesof vascular smooth muscle cells are useful for selecting (e.g., bycloning) or constructing (e.g., by genetic engineering or chemicalsynthesis) appropriately specific vascular smooth muscle bindingproteins. Particularly useful “targets” are internalized by smoothmuscle cells, e.g., as membrane constituent antigen turnover occurs inrenewal. Internalization by cells may also be by mechanisms involvingphagolysosomes, clathrin-coated pits, receptor-mediated redistributionor endocytosis and the like. In a preferred embodiment, such a “target”is exemplified by chondroitin sulfate proteoglycans (CSPGs) synthesizedby vascular smooth muscle cells and pericytes, and a discrete portion(termed an epitope herein) of the CSPG molecule having an apparentmolecular weight of about 250 kD is especially preferred. The 250 kDtarget is an N-linked glycoprotein that is a component of a larger 400kD proteoglycan complex (14). In one presently preferred embodiment ofthe invention, a vascular smooth muscle binding protein is provided byNR-AN-01 monoclonal antibody (a subculture of NR-ML-05) that binds to anepitope in a vascular smooth muscle CSPG target molecule. The monoclonalantibody designated NR-ML-05 reportedly binds a 250 kD CSPG synthesizedby melanoma cells (Morgan et al., U.S. Pat. No. 4,897,255). Smoothmuscle cells and pericytes also reportedly synthesize a 250 kD CSPG aswell as other CSPGs (11). NR-ML-05 binding to smooth muscle cells hasbeen disclosed (Fritzberg et al., U.S. Pat. No. 4,879,225). Monoclonalantibody NR-ML-05 and subculture NR-ML-05 No. 85-41-41-A2, freeze #A2106, have both been deposited with the American Type CultureCollection, Rockville, Md. and granted Accession Nos. HB-5350 andHB-9350, respectively. NR-ML-05 is the parent of, and structurally andfunctionally equivalent to, subclone NR-AN-01, disclosed herein. It willbe recognized that NR-AN-01 is just one example of a vascular smoothmuscle binding protein that specifically associates with the 400 kD CSPGtarget, and that other binding proteins associating with this target andother epitopes in this target (14) are also useful in the therapeuticconjugates and methods of the invention. In the present case, six othermurine monoclonal antibodies and two human chimeric monoclonalantibodies have also been selected, as described herein, thatspecifically target to the 250 kD CSPG of vascular smooth muscle cells.The antibodies also appear to be internalized by the smooth muscle cellsfollowing binding to the cell membrane. Immunoreactivity studies havealso shown the binding of the murine MAbs to the 250 kD antigen in 45human normal tissues and 30 different neoplasms and some of theseresults have been disclosed previously (U.S. Pat. No. 4,879,225). Inthis disclosure and other human clinical studies, MAbs directed to theCSPG 250 kD antigen localized to vascular smooth muscle cells in vivo.Further, it will be recognized that the amino acid residues involved inthe multi-point kinetic association of the NR-AN-01 monoclonal antibodywith a CSPG marker antigenic epitope (i.e., the amino acids constitutingthe complementarity determining regions) are determined bycomputer-assisted molecular modeling and by the use of mutants havingaltered antibody binding affinity. The binding-site amino acids andthree dimensional model of the NR-AN-01 antigen binding site serve as amolecular model for constructing functional equivalents, e.g., shortpolypeptides (“minimal polypeptides”), that have binding affinity for aCSPG synthesized by vascular smooth muscle cells and pericytes.

In a presently preferred embodiment for treating stenosis followingvascular surgical procedures, e.g., PTCA, selected binding proteins,e.g., antibodies or fragments, for use in the practice of the inventionhave a binding affinity of >10⁴ liter/mole for the vascular smoothmuscle 250 kD CSPG, and also the ability to be bound to and internalizedby smooth muscle cells or pericytes.

Three-dimensional modeling is also useful to construct other functionalequivalents that mimic the binding of NR-AN-01 to its antigenic epitope,e.g., “mimetic” chemical compounds that mimic the three-dimensionalaspects of NR-AN-01 binding to its epitope in a CSPG target antigen. Asused herein, “minimal polypeptide” refers to an amino acid sequence ofat least six amino acids in length. As used herein, the term “mimetic”refers to an organic chemical polymer constructed to achieve the properspacing for binding to the amino acids of, for example, an NR-AN-01 CSPGtarget synthesized by vascular smooth muscle cells or pericytes.

It will be recognized that the inventors also contemplate the utility ofhuman monoclonal antibodies or “humanized” murine antibody as a vascularsmooth muscle binding protein in the therapeutic conjugates of theirinvention. For example, murine monoclonal antibody may be “chimerized”by genetically recombining the nucleotide sequence encoding the murineFv region (i.e., containing the antigen binding sites) with thenucleotide sequence encoding a human constant domain region and an Feregion, e.g., in a manner similar to that disclosed in European PatentApplication No. 0,411,893 A2. Some murine residues may also be retainedwithin the human variable region framework domains to ensure propertarget site binding characteristics. Humanized vascular smooth musclebinding partners will be recognized to have the advantage of decreasingthe immunoreactivity of the antibody or polypeptide in the hostrecipient, which may thereby be useful for increasing the in vivohalf-life and reducing the possibility of adverse immune reactions.

Also contemplated as useful binding peptides for restenosis treatmentsustained release dosage forms of the present invention are those thatlocalize to intercellular stroma and matrix located between and amongvascular smooth muscle cells. Such binding peptides deliver thetherapeutic agent to the interstitial space between the target cells.The therapeutic agent is released into such interstitial spaces forsubsequent uptake by the vascular smooth muscle cells. Preferred bindingpeptides of this type are associated with epitopes on collagen,extracellular glycoproteins such as tenascin, reticulum and elasticfibers, cytokeratin and other intercellular matrix components. Minimalpeptides, mimetic organic chemical compounds, human or humanizedmonoclonal antibodies and the like that localize to intracellular stromaand matrix are also useful as binding peptides in this embodiment of thepresent invention. Such binding peptides may be identified andconstructed or isolated in accordance with known techniques. Inpreferred embodiments of the present invention, the interstitial matrixbinding protein binds to a target epitope with an association constantof at least about 10⁻⁴ M.

Useful binding peptides for cancer treatment embodiments of the presentinvention include those associated with cell membrane and cytoplasmicepitopes of cancer cells and the like. These binding peptides localizeto the surface membrane of intact cells and internal epitopes ofdisrupted cells, respectively, and deliver the therapeutic agent forassimilation into the target cells. Minimal peptides, mimetic organiccompounds and human or humanized antibodies that localize to therequisite tumor cell types are also useful as binding peptides of thepresent invention. Such binding peptides may be identified andconstructed or isolated in accordance with known techniques. Preferredbinding peptides of these embodiments of the present invention bind to atarget epitope with an association constant of at least about 10⁻⁶ M.

Binding peptides to membrane and cytoplasmic epitopes and the like thatlocalize to immune system-mediated disease effector cells, e.g., cellsof the lymphoreticular system, are also useful to deliver sustainedrelease dosage forms of the present invention. The therapeutic agent isdelivered to target cells for internalization therein by such sustainedrelease dosage forms. Minimal peptides, mimetic organic compounds andhuman or humanized antibodies that localize to the requisite effectorcell types are also useful as binding peptides of the present invention.Such binding peptides may be identified and constructed or isolated inaccordance with known techniques. Preferred binding peptides of theseembodiments of the present invention bind to a target epitope with anassociation constant of at least about 10⁻⁶ M.

Other preferred binding proteins or peptides useful in the practice ofthe present invention include moieties capable of localizing topathologically proliferating normal tissues, such as pericytes of theintraocular vasculature implicated in degenerative eye disease. Thetherapeutic agent is delivered to target cells for internalizationtherein by such sustained release dosage forms. Minimal peptides,mimetic organic compounds and human or humanized antibodies thatlocalize to the requisite pathologically proliferating normal cell typesare also useful as binding peptides of the present invention. Suchbinding peptides may be identified and constructed or isolated inaccordance with known techniques. Preferred binding peptides of theseembodiments of the present invention bind to a target epitope with anassociation constant of at least about 10⁻⁶ M.

Representative “coupling” methods for linking the therapeutic agentthrough covalent or non-covalent bonds to the vascular smooth musclebinding protein include chemical cross-linkers and heterobifunctionalcross-linking compounds (i.e., “linkers”) that react to form a bondbetween reactive groups (such as hydroxyl, amino, amido, or sulfhydrylgroups) in a therapeutic agent and other reactive groups (of a similarnature) in the vascular smooth muscle binding protein. This bond may be,for example, a peptide bond, disulfide bond, thioester bond, amide bond,thioether bond, and the like. In one illustrative example, conjugates ofmonoclonal antibodies with drugs have been summarized by Morgan and Foon(Monoclonal Antibody Therapy to Cancer: Preclinical Models andInvestigations, Basic and Clinical Tumor Immunology, Vol. 2, KluwerAcademic Publishers, Hingham, Mass.) and by Uhr J. of Immunol.133:i-vii, 1984). In another illustrative example where the conjugatecontains a radionuclide cytostatic agent, U.S. Pat. No. 4,897,255,Fritzberg et al., incorporated herein by reference, is instructive ofcoupling methods that may be useful. In one presently preferredembodiment, the therapeutic conjugate contains a vascular smooth musclebinding protein coupled covalently to a trichothecene drug. In thiscase, the covalent bond of the linkage may be formed between one or moreamino, sulfhydryl, or carboxyl groups of the vascular smooth musclebinding protein and a) the trichothecene itself; b) a trichothecenehemisuccinate carboxylic acid; c) a trichothecene hemisuccinate (HS)N-hydroxy succinimidate ester; or d) trichothecene complexes withpoly-L-lysine or any polymeric carrier. Representative examples ofcoupling methods for preparing therapeutic conjugates containing atrichothecene therapeutic agent are described in U.S. Pat. Nos.4,906,452 and 4,744,981, incorporated herein by reference. Otherexamples using a hydrazide for forming a Schiff base linkage betweenbinding proteins and trichothecenes are disclosed in pending U.S. patentapplication Ser. No. 07/415,154, incorporated herein by reference.

The choice of coupling method will be influenced by the choice ofvascular smooth muscle binding protein or peptide, interstitial matrixbinding protein or peptide and therapeutic agent, and also by suchphysical properties as, e.g., shelf life stability, and/or by suchbiological properties as, e.g., half-life in cells and blood,intracellular compartmentalization route, and the like. For example, inone presently preferred therapeutic conjugate, hemisuccinate conjugatesof the Roridin A therapeutic agent have a longer serum half-life thanthose of Verrucarin A, and this increased stability results in asignificantly increased biological activity.

The sustained release embodiment of the present invention includes atherapeutic agent dispersed within a non-biodegradable or biodegradablepolymeric structure. Such dispersion is conducted in accordance with theprocedure described by Cowsar et al., “Poly(Lactide-Co-Glycolide)Microcapsules for Controlled Release of Steroids,” Methods Enzymology,112:101-116, 1985; Eldridge et al., “Biodegradable and BiocompatiblePoly(DL-Lactide-Co-Glycolide) Microspheres as an Adjuvant forStaphylococcal Enterotoxin B Toxoid Which Enhances the Level ofToxin-Neutralizing Antibodies,” Infection and Immunity, 59:2978-2986,1991; Cohen et al., “Controlled Delivery Systems for Proteins Based onPoly(Lactic/Glycolic Acid) Microspheres,” Pharmaceutical Research,8(6):713-720, 1991; and Sanders et al., “Controlled Release of aLuteinizing Hormone-Releasing Hormone Analogue fromPoly(D,L-Lactide-Co-Glycolide) Microspheres,” J. Pharmaceutical Science,73(9):1294-1297, 1984.

The physical and chemical character of the sustained release dosage formof the present invention is amenable to several alternative modes ofattachment to binding proteins or peptides. Dosage forms (sustainedrelease-type) of the present invention are capable of binding to bindingproteins/peptides through, for example, covalent linkages, intermediateligand sandwich attachment, or non-covalent adsorption or partialentrapment. When the preferred poly-lactic/glycolic acid particulatesare formed with the therapeutic agent dispersed therein, the unchargedpolymer backbone is oriented both inward (with the quasi lipophilictherapeutic agent contained therein) and outward along with a majorityof the terminal carboxy groups. These surface carboxy groups may serveas covalent attachment sites when activated by, for example, acarbodiimide for nucleophilic groups of the binding protein/peptide.Such nucleophilic groups include lysine epsilon amino groups (amidelinkage), serine hydroxyl groups (ester linkage) or cysteine mercaptangroups (thioester linkage). Reactions with particular groups depend uponpH and the reduction state of the reaction conditions.

For example, poly-lactic/glycolic acid particulates having terminalcarboxylic acid groups are reacted with N-hydroxybenztriazole in thepresence of a water soluble carbodiimide of the formula R—N═C═N—R′(wherein R is a 3-dimethylaminopropyl group or the like and R′ is anethyl group or the like). The benztriazole-derivatized particulates(i.e., activated imidate-bearing moieties) are then reacted with aprotein/peptide nucleophilic moiety such as an available epsilon aminomoiety. Alternatively, p-nitrophenol, tetrafluorophenol,N-hydroxysuccinimide or like molecules are useful to form an activeester with the terminal carboxy groups of poly-lactic/glycolic acidparticulates in the presence of carbodiimide. Other bindingprotein/peptide nucleophilic moieties include hydroxyl groups of serine,endogenous free thiols of cysteine, thiol groups resulting fromreduction of binding protein/peptide disulfide bridges using reducingagents commonly employed for that purpose (e.g., cysteine,dithiothreitol, mercaptoethanol and the like) and the like.Additionally, the terminal carboxy groups of the polylactic/glycolicacid particulates are activatable by reaction with thionyl chloride toform an acyl chloride derivatized moiety. The derivatized particulatesare then reacted with binding peptide/protein nucleophilic groups toform targeted dosage forms of the present invention.

Direct sustained release dosage form-binding protein or peptideconjugation may disrupt binding protein/peptide target cell recognition.Ligand sandwich attachment techniques are useful alternatives to achievesustained release dosage form-binding protein/peptide attachment. Suchtechniques involve the formation of a primary peptide or protein shellusing a protein that does not bind to the target cell population.Binding protein/peptide is then bound to the primary peptide or proteinshell to provide the resultant particulate with functional bindingprotein/peptide. An exemplary ligand sandwich approach involves covalentattachment of avidin or streptavidin to the particulates throughfunctional groups as described above with respect to the “direct”binding approach. The binding protein or peptide is derivatized,preferably minimally, with functionalized biotin (e.g., through activeester, hydrazide, iodoacetal, maleimidyl or like functional groups).Ligand (i.e., binding peptide or protein/functionalized biotin)attachment to the available biotin binding sites of theavidin/streptavidin primary protein shell occurs through the use of asaturating amount of biotinylated protein/peptide.

For example, poly-lactic/glycolic acid particulates having terminalcarboxylic acid groups are activated with carbodiimide and subsequentlyreacted with avidin or streptavidin. The binding protein or peptide isreacted with biotinamidocaproate N-hydroxysuccinimide ester at a 1-3molar offering of biotin-containing compound to the bindingprotein/peptide to form a biotinylated binding protein/peptide. A molarexcess of the biotinylated binding protein/peptide is incubated with theavidin-derivatized particulates to form a targeted dosage form of thepresent invention.

Alternatively, the particulate carboxy groups are biotinylated (e.g.,through carbodiimide activation of the carboxy group and subsequentreaction with amino alkyl biotinamide). The biotinylated particulatesare then incubated with a saturating concentration (i.e., a molarexcess) of avidin or streptavidin to form protein coated particulateshaving free biotin binding sites. Such coated particulates are thencapable of reaction with a molar excess of biotinylated binding proteinformed as described above. Another option involves avidin orstreptavidin bound binding peptide or protein attachment to biotinylatedparticulates.

In addition, binding protein/peptide-particulate attachment can beachieved by adsorption of the binding peptide to the particulate,resulting from the nonionic character of the partially exposed polymerbackbone of the particulate. Under high ionic strength conditions (e.g.,1.0 molar NaCl), hydrogen and hydrophobic particulate-bindingprotein/peptide binding are favored.

Moreover, binding protein/peptide may be partially entrapped in theparticulate polymeric matrix upon formation thereof. Under thesecircumstances, such entrapped binding protein/peptide provides residualselective binding character to the particulate. Mild particulateformation conditions, such as those employed by Cohen et al.,Pharmaceutical Research, 8: 713-720 (1991), are preferred for thisembodiment of the present invention. Such entrapped binding protein isalso useful in target cell reattachment of a partially degradedparticulate that has undergone exocytosis. Other polymeric particulatedosage forms (e.g., non-biodegradable dosage forms) having differentexposed functional groups can be bound to binding proteins or peptidesin accordance with the principles discussed above.

Exemplary non-biodegradable polymers useful in the practice of thepresent invention are polystyrenes, polypropylenes, styrene acryliccopolymers and the like. Such non-biodegradable polymers incorporate orcan be derivatized to incorporate functional groups for attachment ofbinding protein/peptide, including carboxylic acid groups, aliphaticprimary amino groups, aromatic amino groups and hydroxyl groups.

Carboxylic acid functional groups are coupled to binding protein orpeptide using, for example, the reaction mechanisms set forth above forpoly-lactic/glycolic acid biodegradable polymeric particulate dosageforms. Primary amino functional groups are coupled by, for example,reaction thereof with succinic anhydride to form a terminal carboxymoiety that can be bound to binding peptide/protein as described above.Additionally, primary amino groups can be activated with cyanogenbromide and form guanidine linkages with binding protein/peptide primaryamino groups. Aromatic amino functional groups are, for example,diazotized with nitrous acid to form diazonium moieties which react withbinding protein/peptide tyrosines, thereby producing a diazo bondbetween the non-biodegradable particulate and the bindingprotein/peptide. Hydroxyl functional groups are coupled to bindingprotein/peptide primary amino groups by, for example, converting thehydroxyl moiety to a terminal carboxylic acid functional group. Such aconversion can be accomplished through reaction with chloroacetic acidfollowed by reaction with carbodiimide. Sandwich, adsorption andentrapment techniques, discussed above with respect to biodegradableparticulates, are analogously applicable to non-biodegradableparticulate-binding protein/peptide affixation.

In a preferred embodiment, targeting is specific for potentiallyproliferating cells that result in increased smooth muscle in theintimal region of a traumatized vascular site, e.g., followingangioplasty, e.g., pericytes and vascular smooth muscle cells. Aspectsof the invention relate to therapeutic modalities in which thetherapeutic conjugate of the invention is used to delay, reduce, oreliminate smooth muscle proliferation after angioplasty, e.g., PTCA,atheroectomy and percutaneous transluminal coronary rotationalatheroblation.

In another preferred embodiment, targeting is specific for primary ormetastatic tumor foci accessible to local administration, e.g., tumorsexposed for infiltration by laparotomy or visible for fluoroscopic orcomputerized tomography guiding and infusion needle administration tointernal tumor foci or tumors confined to a small area or cavity withinthe mammal, e.g., ovarian cancer located in the abdomen, focal ormultifocal liver tumors or the like. Aspects of this embodiment of theinvention involve therapeutical modalities wherein the therapeutic agentis cytotoxic to the target cells or metabolically modulates the cells,increasing their sensitivity to chemotherapy and/or radiation therapy.

In another embodiment, targeting is specific for a local administrationaccessible effector cell population implicated in immune system-mediateddiseases, e.g., arthritis, intraocular immune system-mediated disease orsprue. Aspects of this embodiment of the present invention involvetherapeutic modalities wherein the therapeutic agent is cytotoxic ormodifies the biological response of the target cells to effect atherapeutic objective.

In another embodiment, targeting is specific for a local administrationaccessible pathologically proliferating or hyperactive normal cellpopulation implicated in, e.g., degenerative eye disease, cornealpannus, hyperactive endocrine glands or the like. Aspects of thisembodiment of the present invention involve therapeutic modalitieswherein the therapeutic agent reduces or eliminates proliferation orhyperactivity of the target cell population.

For treatment of a traumatized or diseased vascular site, thetherapeutic conjugates or dosage forms of the invention may beadministered to the host using an infusion catheter, such as produced byC. R. Bard Inc., Billerica, Mass., or that disclosed by Wolinsky (7;U.S. Pat. No. 4,824,436) or Spears (U.S. Pat. No. 4,512,762). In thiscase, a therapeutically effective dosage of the therapeutic conjugatewill be typically reached when the concentration of conjugate in thefluid space between the balloons of the catheter is in the range ofabout 10⁻³ to 10⁻¹² M. It will be recognized from the Examples providedherewith that therapeutic conjugates of the invention may only need tobe delivered in an anti-proliferative therapeutic dosage sufficient toexpose the proximal (6 to 9) cell layers of the intimal or tunica mediacells lining the lumen to the therapeutic anti-proliferative conjugate,whereas the anti-contractile therapeutic dosage needs to expose theentire tunica media, and further that this dosage can be determinedempirically, e.g., by a) infusing vessels from suitable animal modelsystems and using immunohistochemical methods to detect the conjugateand its effects (e.g., such as exemplified in the EXAMPLES below); andb) conducting suitable in vitro studies such as exemplified in EXAMPLES3, 4, and 5, below.

In a representative example, this therapeutically effective dosage isachieved by preparing 10 ml of a 200 μg/ml therapeutic conjugatesolution, wherein the vascular smooth muscle protein binding protein isNR-AN-01 and the therapeutic agent is Roridin A, a trichothecene drug.For treating vascular trauma, e.g., resulting from surgery or disease(e.g., see below), when the therapeutic conjugate is administered withan infusion catheter, 10 ml will commonly be sufficient volume to fillthe catheter and infuse 1 to 1.5 ml into one to three traumatic lesionsites in the vessel wall. It will be recognized by those skilled in theart that desired therapeutically effective dosages of a therapeuticconjugate according to the invention will be dependent on severalfactors, including, e.g.: a) the binding affinity of the vascular smoothmuscle binding protein in the therapeutic conjugate; b) the atmosphericpressure applied during infusion; c) the time over which the therapeuticconjugate administered resides at the vascular site; d) the nature ofthe therapeutic agent employed; and/or e) the nature of the vasculartrauma and therapy desired. Those skilled practitioners trained todeliver drugs at therapeutically effective dosages (e.g., by monitoringdrug levels and observing clinical effects in patients) will determinethe optimal dosage for an individual patient based on experience andprofessional judgment. In a preferred embodiment, about 0.3 atm (i.e.,300 mm of Hg) to about 5 atm of pressure applied for 15 seconds to 3minutes directly to the vascular wall is adequate to achieveinfiltration of a therapeutic conjugate containing the NR-AN-01 bindingprotein into the smooth muscle layers of a mammalian artery wall. Thoseskilled in the art will recognize that infiltration of the therapeuticconjugate into intimal layers of a diseased human vessel wall willprobably be variable and will need to be determined on an individualbasis.

Sustained release dosage forms of an embodiment of the invention mayonly need to be delivered in an anti-proliferative therapeutic dosagesufficient to expose the proximal (6 to 9) cell layers of the tunicamedia smooth muscle cells lining the lumen to the dosage form. Thisdosage is determinable empirically, e.g., by a) infusing vessels fromsuitable animal model systems and using immunohistochemical, fluorescentor electron microscopy methods to detect the dosage form and itseffects; and b) conducting suitable in vitro studies.

In a representative example, this therapeutically effective dosage isachieved by determining in smooth muscle cell tissue culture thepericellular agent dosage, which at a continuous exposure results in atherapeutic effect between the toxic and minimal effective doses. Thistherapeutic level is obtained in vivo by determining the size, numberand therapeutic agent concentration and release rate required forparticulates infused between the smooth muscle cells of the artery wallto maintain this pericellular therapeutic dosage. The dosage form shouldrelease the therapeutic agent at a rate that approximates thepericellular dose of the following exemplary therapeutic agents: fromabout 0.01 to about 100 micrograms/ml nitroglycerin, from about 1.0 toabout 1000 micrograms/ml of suramin, from about 0.001 to about 100micrograms/ml for cytochalasin, and from about 0.01 to about 10⁵nanograms/ml of staurosporin as well as from about 0.001 to about 100micrograms/ml taxol.

For TGF-beta activators or production stimulators, such as tamoxifen,several exemplary dosing regimens are contemplated, depending upon thecondition being treated and the stage to which the condition hasprogressed. For prophylactic purposes with respect to atherosclerosis,for example, a low chronic dose sufficient to elevate in vivo TGF-betaproduction or activation is contemplated. An exemplary dose of this typeis about 0.1 mg/kg/day (ranging between about 0.1 and about 10mg/kg/day). Another exemplary dose range is from about 0.01 to about1000 micrograms/ml. Low doses, such as 0.1 ng/Kg/day, are alsocontemplated for use with respect to ameliorating stenosis followingrelatively low trauma injury or intervention, such as vein grafts ortransplants or organ allografts, for example. No adverse side effects(e.g., nausea as experienced by recipients of higher doseadministrations when tamoxifen has been employed in the treatment ofbreast cancer) are anticipated with respect to these chronic or lowdosing regimens.

For prevention of restenosis following angioplasty, an example of ahigher trauma injury or intervention resulting in a stronger acuteproliferative stimulus to smooth muscle cells, a higher dose would berequired. For example, a dosing regimen is contemplated which involves asingle “pre-loading” dose (or multiple, smaller pre-loading doses) givenbefore or at the time of the intervention, with a chronic smaller(follow up) dose delivered for two to three weeks or longer followingintervention. For example, a single pre-loading dose may be administeredabout 24 hours prior to intervention, while multiple preloading dosesmay be administered daily for several days prior to intervention. Anexemplary single pre-loading dose is about 50 mg/kg (ranging betweenabout 5 and about 1000 mg/kg), while an exemplary multiple pre-loadingindividual dose is about 10 mg/kg/day (ranging between about 0.01 and 10mg/kg/day). Such a dosing regimen may involve a systemic pre-loadingdose followed by a sustained release chronic dose, or the sustainedrelease dosage form may be designed to deliver a large dose over a shorttime interval as well as a smaller chronic dose for the desired timeperiod thereafter. Some nausea may be encountered at the higher dose;however, the use of a sustained release or other targeted dosage form isexpected to obviate this side effect, because the recipient will not besubjected to a high systemic dose of the therapeutic agent.

It will be recognized by those skilled in the art that desiredtherapeutically effective dosages of the catheter administered sustainedrelease dosage forms of the invention will be dependent on severalfactors, including, e.g.: a) the binding affinity of the binding proteinassociated with the dosage form, if any; b) the atmospheric pressure andduration of the infusion; c) the time over which the dosage formadministered resides at the target site; d) the rate of therapeuticagent release from the particulate dosage form; e) the nature of thetherapeutic agent employed; f) the nature of the trauma and/or therapydesired; and/or g) the intercellular and/or intracellular localizationof the particulate dosage form. Those skilled practitioners trained todeliver drugs at therapeutically effective dosages, (e.g., by monitoringtherapeutic agent levels and observing clinical effects in patients) arecapable of determining the optimal dosage for an individual patientbased on experience and professional judgment. In a preferredembodiment, about 0.3 atm (i.e., 300 mm of Hg) to about 3 atm ofpressure applied for 15 seconds to 3 minutes to the arterial wall isadequate to achieve infiltration of a sustained release dosage formbound to the NR-AN-01 binding protein into the smooth muscle layers of amammalian artery wall. Wolinsky et al., “Direct Intraarterial WallInjection of Microparticles Via a Catheter: A Potential Drug DeliveryStrategy Following Angioplasty,” Am. Heart Jour., 122(4):1136-1140,1991. Those skilled in the art will recognize that infiltration of asustained release dosage form into a target cell population willprobably be variable and will need to be determined on an individualbasis.

It will also be recognized that the selection of a therapeutic agentthat exerts its effects intracellularly, e.g., on ribosomes or DNAmetabolism, will influence the dosage and time required to achieve atherapeutically effective dosage, and that this process can be modeledin vitro and in animal studies, such as those described in the Examplesprovided below, to find the range of concentrations over which thetherapeutic conjugate or dosage form should be administered to achieveits effects of delaying, reducing or preventing restenosis followingangioplasty. For example, therapeutic conjugates radiolabeled withalpha-, beta- or gamma-emitters of known specific activities (e.g.,millicuries per millimole or milligram of protein) are useful fordetermining the therapeutically effective dosage by using them in animalstudies and human trials with quantitative imaging or autoradiography ofhistological tissue sections to determine the concentration oftherapeutic conjugate that is required by the therapeutic protocol. Atherapeutically effective dosage of the therapeutic conjugate or dosageform will be reached when at least three conditions are met: namely, (1)the therapeutic conjugate or dosage form is distributed in the intimallayers of the traumatically injured vessel; (2) the therapeuticconjugate or dosage form is distributed within the desired intracellularcompartment of the smooth muscle cells, i.e., that compartment necessaryfor the action of the therapeutic agent, or the therapeutic agentreleased from the dosage form extracellularly is distributed within therelevant intracellular compartment; and (3) the therapeutic agentinhibits the desired cellular activity of the vascular smooth musclecell, e.g., proliferation, migration, increased cellular volume, matrixsynthesis, cell contraction and the like described above.

It will be recognized that where the therapeutic conjugate or dosageform is to be delivered with an infusion catheter, the therapeuticdosage required to achieve the desired inhibitory activity for atherapeutic conjugate or dosage form can also be anticipated through theuse of in vitro studies. In a preferred aspect, the infusion cathetermay be conveniently a double balloon or quadruple balloon catheter witha permeable membrane. In one representative embodiment, atherapeutically effective dosage of a therapeutic conjugate or dosageform is useful in treating vascular trauma resulting from disease (e.g.,atherosclerosis, aneurysm, or the like) or vascular surgical proceduressuch as angioplasty, atheroectomy, placement of a stent (e.g., in avessel), thrombectomy, and grafting. Atheroectomy may be performed, forexample, by surgical excision, ultrasound or laser treatment, or by highpressure fluid flow. Grafting may be, for example, vascular graftingusing natural or synthetic materials or surgical anastomosis of vesselssuch as, e.g., during organ grafting. Those skilled in the art willrecognize that the appropriate therapeutic dosage for a given vascularsurgical procedure (above) is determined in in vitro and in vivo animalmodel studies, and in human preclinical trials. In the EXAMPLES providedbelow, a therapeutic conjugate containing Roridin A and NR-AN-01achieved a therapeutically effective dosage in vivo at a concentrationwhich inhibited cellular protein synthesis in test cells in vitro by atleast 5 to 50%, as judged by incorporation of radiolabeled amino acids.

In the case of therapeutic agents of conjugates or dosage formscontaining anti-migratory or anti-matrix therapeutic agents, cellmigration and cell adherence in in vitro assays, respectively, may beused for determining the concentration at which a therapeuticallyeffective dosage will be reached in the fluid space created by theinfusion catheter in the vessel wall.

While one representative embodiment of the invention relates totherapeutic methods employing an infusion catheter, it will berecognized that other methods for drug delivery or routes ofadministration may also be useful, e.g., injection by the intravenous,intralymphatic, intrathecal, intraarterial, local delivery by implantedosmotic pumps or other intracavity routes. Dosage form administration bythese routes in accordance with the present invention may be continuousor intermittent, depending, for example, upon the patient'sphysiological condition, whether the purpose of the administration istherapeutic or prophylactic and other factors known to and evaluable bya skilled practitioner. For intravenous administration, nanoparticulatedosage forms of the present invention are preferred. Intravenousadministration of nanoparticulates is useful, for example, wherevascular permeability is increased in tumors for leakage, especially innecrotic areas of tumors having damaged vessels which allow the leakageof particles into the interstitial fluid, and where artery walls havebeen denuded and traumatized allowing the particles to enter theinterstitial area of the tunica media.

In the practice of certain embodiments of the present invention,catheter and other administration routes are preferably conducted usingdosage forms or therapeutic agents dispersed in a pharmaceuticallyacceptable carrier that is in liquid phase. Useful pharmaceuticallyacceptable carriers for these purposes include generally employedcarriers, such as phosphate buffered saline solution, water, emulsions(e.g., oil/water and water/oil emulsions) and wetting agents of varioustypes.

Advantageously, non-coupled vascular smooth muscle cell binding protein(e.g., free NR-AN-01 antibody) is administered prior to administrationof the therapeutic agent conjugate or dosage form to provide a blockerof non-specific binding to cross-reactive sites. Blocking of such sitesis important because vascular smooth muscle cell binding proteins willgenerally have some low level of cross-reactivity with cells in tissuesother than the desired smooth muscle cells. Such blocking can improvelocalization of the therapeutic conjugate or dosage form at the specificvascular site, e.g., by making more of the therapeutic conjugateavailable to the cells. As an example, non-coupled vascular smoothmuscle binding protein is administered from about 5 minutes to about 48hours, most preferably from about 5 minutes to about 30 minutes, priorto administration of the therapeutic conjugate or dosage form. In onepreferred embodiment of the invention, the unlabeled specific “blocker”is a monovalent or bivalent form of an antibody (e.g., a whole antibodyor an F(ab)′₂, Fab, Fab′, or Fv fragment of an antibody). The monovalentform of the antibody has the advantage of minimizing displacement of thetherapeutic conjugate or dosage form while maximizing blocking of thenon-specific cross-reactive sites. The non-coupled vascular smoothmuscle cell binding protein is administered in an amount effective toblocking binding of a least a portion of the non-specific cross-reactivesites in a patient. The amount may vary according to such factors as theweight of the patient and the nature of the binding protein. In general,about 0.06 mg to 0.20 mg per kg body weight or more of the unlabeledspecific blocker is administered to a human.

In addition, a second irrelevant vascular smooth muscle cell bindingprotein may optionally be administered to a patient prior toadministration of the therapeutic conjugate or dosage form to reducenon-specific binding of the therapeutic conjugate or dosage form totissues. In a preferred embodiment, the irrelevant binding protein maybe an antibody which does not bind to sites in the patient throughantigen-specific binding, but instead binds in a non-specific manner,e.g., through Fc receptor binding reticuloendothelial cells,asialo-receptor binding, and by binding to ubiquitin-expressing cells.The irrelevant “blocker” decreases non-specific binding of thetherapeutic conjugate or dosage form and thus reduces side-effects,e.g., tissue toxicity, associated with the use of the therapeuticconjugate or dosage form. The irrelevant “blocker” is advantageouslyadministered from 5 minutes to 48 hours, most preferably from 15 minutesto one hour, prior to administration of the therapeutic conjugate ordosage form, although the length of time may vary depending upon thetherapeutic conjugate and route or method of injection. Representativeexamples of irrelevant “blockers” include antibodies that arenonreactive with human tissues and receptors or cellular and serumproteins prepared from animal sources that when tested are found not tobind in a specific manner (e.g., with a Ka<10³ M⁻¹) to human cellmembrane targets.

It will be recognized that the conjugates and dosage forms of theinvention are not restricted in use for therapy following angioplasty;rather, the usefulness of the therapeutic conjugates and dosage formswill be proscribed by their ability to inhibit cellular activities ofsmooth muscle cells and pericytes in the vascular wall. Thus, otheraspects of the invention include therapeutic conjugates and dosage formsand protocols useful in early therapeutic intervention for reducing,delaying, or eliminating (and even reversing) atherosclerotic plaquesand areas of vascular wall hypertrophy and/or hyperplasia. Therapeuticconjugates and dosage forms of the invention also find utility for earlyintervention in pre-atherosclerotic conditions, e.g., they are useful inpatients at a high risk of developing atherosclerosis or with signs ofhypertension resulting from atherosclerotic changes in vessels or vesselstenosis due to hypertrophy of the vessel wall.

The therapeutic conjugates and dosage forms of the invention may also beused in therapeutic modalities for enhancing the regrowth of endothelialcells in injured vascular tissues and in many kinds of wound sitesincluding epithelial wounds. In these therapeutic modalities, thetherapeutic conjugates and dosage forms of the invention find utility ininhibiting the migration and/or proliferation of smooth muscle cells orpericytes. Smooth muscle cells and pericytes have been implicated in theproduction of factors in vitro that inhibit endothelial cellproliferation, and their proliferation can also result in a physicalbarrier to establishing a continuous endothelium. Thus, the therapeuticconjugates and dosage forms of the invention find utility in promotingneo-angiogenesis and increased re-endothelialization, e.g., during woundhealing, vessel grafts and following vascular surgery. The dosage formsmay also release therapeutic modalities that stimulate or speed upre-endothelialization of the damaged vessel wall. An exemplarytherapeutic agent for this purpose is vascular permeability factor.

Still other aspects of the invention relate to therapeutic modalitiesfor enhancing wound healing in a vascular site and improving thestructural and elastic properties of healed vascular tissues. In thesetherapeutic modalities using the therapeutic conjugate or dosage form ofthe invention, i.e., to inhibit the migration and proliferation ofsmooth muscle cells or pericytes in a vessel wall, the strength andquality of healing of the vessel wall are improved. Smooth muscle cellsin the vascular wound site contribute to the normal process ofcontraction of the wound site which promotes wound healing. It ispresently believed that migration and proliferation of smooth musclecells and matrix secretion by transformed smooth muscle cells maydetract from this normal process and impair the long-term structural andelastic qualities of the healed vessel. Thus, other aspects of theinvention provide for therapeutic conjugates and dosage forms thatinhibit smooth muscle and pericyte proliferation and migration as wellas morphological transformation, and improve the quality of the healedvasculature.

The present invention also provides a combination therapeutic methodinvolving a cytocidal therapeutic conjugate and a cytostatic therapeuticagent. The cytocidal conjugate includes a binding partner (such as aprotein or peptide) capable of specifically localizing to vascularsmooth muscle cells and an active agent capable of killing such cells.The cytocidal conjugate is administered, preferably intravenously orthrough any other convenient route therefor, localizes to the targetsmooth muscle cells, and destroys proliferating cells involved instenotic or restenotic events. This cellular destruction causes therelease of mitogens and other metabolic events, which events generallylead, in turn, to vascular smooth muscle cell proliferation. Thesustained release anti-proliferative or anti-contractile dosage forms ofthe present invention are next administered, preferably through aninfusion catheter or any convenient dosage form therefor. The sustainedrelease dosage form retards the vascular smooth muscle cellproliferation and/or migration and contraction, thereby maintainingluminal diameter. This treatment methodology constitutes a biologicalarteromyectomy useful in stenotic vessels resulting from vascular smoothmuscle cell hyperplasia and the like.

Alternatively, a combination protocol can be employed involving a, forexample, systemically administered TGF-beta, TGF-beta activator orTGF-beta production stimulator capable of stabilizing or organizing theproliferation occurring at a diseased or traumatized smooth muscle site.The therapeutic or prophylactic agent combined by, for example, localadministration in protocols employing the aforementionedstabilizer/organizer may be either a cytotoxic agent (e.g., freecytotoxic agent, a cytotoxic conjugate, or a sustained dosage formincorporating a cytotoxic agent) or a cytostatic agent (e.g., free,targeted or sustained release formulations of an agent capable ofgenerating a biological stenting effect, an anti-migratory agent, acytoskeletal inhibitor, a metabolic inhibitor, an anti-proliferativeagent or the like).

When a cytotoxic agent is employed, the stabilizer or organizer ispreferably administered prior to cytotoxic agent administration. Apreferred embodiment of this aspect of the present invention for theprevention or treatment of restenosis features the following steps:

1) systemic administration of a large, prophylactically effective doseof tamoxifen;

2) after the passage of from about 0 to about 72 hours (preferably 24 to72), an effective amount of a, for example, Pseudomonasexotoxin-monoclonal antibody conjugate capable of localizing to vascularsmooth muscle cells is locally administered (e.g., via a catheter duringan angioplasty procedure); and

3) daily system administrations of smaller, follow up doses oftamoxifen. Optionally, a follow up dose of tamoxifen could also belocally administered in step 2.

Using this protocol offers reduced and more highly organized or morestable proliferation by smooth muscles cells that are susceptible to acytotoxic agent targeted thereto. The cytotoxic agent acts on theproliferating cells. The follow up doses of tamoxifen facilitate theprevention of proliferation resulting from smooth muscle cell deathcaused by the action of the cytotoxic agent.

When cytostatic agents are employed, the stabilizer or organizer ispreferably administered prior to cytostatic agent administration. Apreferred embodiment of this aspect of the present invention for theprevention or treatment of restenosis features the following steps:

1) systemic administration of a large, prophylactically effective doseof tamoxifen;

2) after the passage of from about 0 to about 72 hours (preferably 24-72hours), an effective amount of cytochalasin B is locally administered(e.g., via a catheter during an angioplasty procedure); and

3) daily system administrations of smaller, follow up doses oftamoxifen. Optionally, a follow up dose of tamoxifen could also belocally administered in step 2.

Using this protocol offers reduced and more highly organized or morestable proliferation by smooth muscles cells in combination with abiological stenting effect.

The present invention also provides methods for the treatment of cancerand immune system-mediated diseases through local administration of atargeted particulate dosage form. The particulate dosage form is, forexample, administered locally into primary and/or metastatic foci ofcancerous target cells. Local administration is preferably conductedusing an infusion needle or intraluminal administration route, infusingthe particulate dosage form in the intercellular region of the tumortissue or in luminal fluid surrounding the tumor cells.

Primary foci introduction is preferably conducted with respect to targetcells that are generally situated in confined areas within a mammal,e.g., ovarian carcinomas located in the abdominal cavity. The dosageform of the present invention binds to the target cell population and,optionally, is internalized therein for release of the therapeutic agentover time. Local administration of dosage forms of the present inventionto such primary foci results in a localized effect on such target cells,with limited exposure of other sensitive organs, e.g., the bone marrowand kidneys, to the therapeutic agent.

When metastatic foci constitute the target cell population, theadministered microparticles and larger nanoparticles are primarily boundto the target cells situated near the infusion site and are, optionally,internalized for release of the therapeutic agent, thereby generating amarked and localized effect on the target cells immediately surroundingthe infusion site. In addition, smaller nanoparticles followinterstitial fluid flow or lymphatic drainage channels and bind totarget cells that are distal to the infusion site and undergoinglymphatic metastasis.

The targeted dosage forms of this embodiment of the present inventioncan be used in combination with more commonly employed immunoconjugatetherapy. In this manner, the immunoconjugate achieves a systemic effectwithin the limits of systemic toxicity, while the dosage form of thepresent invention delivers a concentrated and sustained dose oftherapeutic agent to the primary and metastatic foci, which oftenreceive an inadequate therapeutic dose from such “systemic”immunoconjugate administration alone, and avoids or minimizes systemictoxic effects.

Where the target cell population can be accessed by localadministration, the dosage forms of the present invention are utilizedto control immune system-mediated diseases. Exemplary of such diseasesare arthritis, sprue, uveitis, endophthalmitis, keratitis and the like.The target cell populations implicated in these embodiments of thepresent invention are confined to a body cavity or space, such as jointcapsules, pleural and abdominal cavity, eye and sub-conjunctival space,respectively. Local administration is preferably conducted using aninfusion needle for a intrapleural, intraperitoneal, intraocular orsub-conjunctival administration route.

This embodiment of the present invention provides a more intense,localized modulation of immune system cells with minimal effect on thesystemic immune system cells. Optionally, the systemic cells of theimmune system are simultaneously treatable with a chemotherapeutic agentconjugated to a binding protein or peptide. Such a conjugate preferablypenetrates from the vascular lumen into target immune system cells.

The local particulate dosage form administration may also localize tonormal tissues that have been stimulated to proliferate, therebyreducing or eliminating such pathological (i.e., hyperactive)conditions. An example of this embodiment of the present inventioninvolves intraocular administration of a particulate dosage form coatedwith a binding protein or peptide that localizes to pericytes and smoothmuscle cells of neovascularizing tissue. Proliferation of thesepericytes causes degenerative eye disease. Preferred dosage forms of thepresent invention release compounds capable of suppressing thepathological proliferation of the target cell population. The preferreddosage forms can also release compounds that increase vessel lumen areaand blood flow, reducing the pathological alterations produced by thisreduced blood supply.

Still another aspect of the present invention relates to therapeuticmodalities for maintaining an expanded luminal volume followingangioplasty or other vessel trauma. One embodiment of this aspect of thepresent invention involves administration of a therapeutic agent capableof inhibiting the ability of vascular smooth muscle cells to contract.Exemplary agents useful in the practice of this aspect of the presentinvention are those capable of causing a traumatized artery to losevascular tone, such that normal vascular hydrostatic pressure (i.e.,blood pressure) expands the flaccid vessel to or near to its maximalphysiological diameter. Loss of vascular tone may be caused by agentsthat interfere with the formation or function of contractile proteins(e.g., actin, myosin, tropomyosin, caldesmon, calponin or the like).This interference can occur directly or indirectly through, for example,inhibition of calcium modulation, phosphorylation or other metabolicpathways implicated in contraction of vascular smooth muscle cells.

Inhibition of cellular contraction (i.e., loss of vascular tone) mayoperate through two mechanisms to reduce the degree of vascularstenosis. First, inhibition of cellular contraction for a prolongedperiod of time limits the number of smooth muscle cells that migratefrom the tunica media into the intima, the thickening of which resultsin vascular luminal stenosis. Second, inhibition of cellular contractioncauses the smooth muscle wall to relax and dilate under normal vascularhydrostatic pressure (i.e., blood pressure). Therapeutic agents, such asthe cytochalasins, inhibit smooth muscle cell contraction withoutabolishing the protein synthesis necessary for traumatized,post-angioplasty or other surgically- or disease-damaged, smooth musclecells to repair themselves. Protein synthesis is also necessary for thesmooth muscle cells to secrete matrix, which fixes or retains the lumenin a state near its maximum systolic diameter as the vascular lesionstabilizes (i.e., a biologically-induced stenting effect).

This biological stenting effect not only results in an expanded vesselluminal area and increased blood flow rate through the vessel, but alsosignificantly reduces elastic recoil following angioplasty. Elasticrecoil is an acute closure of the vessel associated with vasospasm orearly relaxation of the muscular wall, due to trauma shock resultingfrom vessel over-stretching by a balloon catheter during angioplasty.This spasm of the tunica media which leads to decreases in the luminalarea may occur within hours, days or weeks after the balloon dilation,as restoration of vascular muscle wall tone occurs. Recent observationsduring microscopic examination of atheroectomy specimens suggest thatelastic recoil may occur in up to 25% of angioplasty proceduresclassified as successful, based on the initial post-procedure angiogram.Because the biological stenting procedure relaxes the artery wallfollowing balloon angioplasty, the clinician can eliminateover-inflation and its resultant trauma shock as a means to diminish ordelay the vessel spasm or elastic recoil. Reduction or elimination ofover-inflation decreases trauma to the muscular wall of the vessel,thereby reducing the determinants of smooth muscle cell proliferation inthe intima and, therefore, reducing the incidence or severity ofrestenosis.

Biological stenting also decreases the incidence of thrombus formation.In pig femoral arteries treated with cytochalasin B, for example, theincidence of mural microthrombi was decreased as compared to the balloontraumatized arteries that were not treated with the therapeutic agent.This phenomenon appears to be a secondary benefit that may result fromthe increased blood flow through the traumatized vessel, said benefitbeing obtained through the practice of the present invention.

Cytochalasins are exemplary therapeutic agents capable of generating abiological stenting effect on vascular smooth muscle cells.Cytochalasins are thought to inhibit both migration and contraction ofvascular smooth muscle cells by interacting with actin. Thecytochalasins interact with the ends of filamentous actin to inhibit theelongation of the actin filaments. Low doses of cytochalasins (e.g.,cytochalasin B) also disrupt microfilament networks of actin. In vitrodata indicate that after vascular smooth muscle cells clear cytochalasinB, the cells regenerate enough polymerized actin to resume migrationwithin about 24 hours. In vivo assessments reveal that vascular smoothmuscle cells regain vascular tone within 2 to 4 days. It is during thisrecuperative period that the lumen diameter fixation and biologicalstenting effect occurs.

The therapeutic agent may be targeted, but is preferably administereddirectly to the traumatized vessel following the angioplasty or othertraumatic event. The biological stenting effect of cytochalasin B, forexample, is achievable using a single infusion of the therapeutic agentinto the traumatized region of the vessel wall at a dose concentrationranging from about 0.1 microgram/ml to about 1.0 micrograms/ml.

Inhibition of vascular smooth muscle cell migration (from the tunicamedia to the intima) has been demonstrated in the same dose range(Example 11); however, a sustained exposure of the vessel to thetherapeutic agent is preferable in order to maximize theseanti-migratory effects. If the vascular smooth muscle cells cannotmigrate into the intima, they cannot proliferate there. Should vascularsmooth muscle cells migrate to the intima, a subsequently administeredanti-proliferative sustained release dosage form inhibits the intimalproliferation. As a result, the sustained release dosage form of thepresent invention, incorporating a cytochalasin or otheranti-proliferative therapeutic agent, can be administered in combinationwith a free cytochalasin therapeutic agent. In this manner, thebiological stenting effect, as well as an anti-proliferative oranti-migratory effect, can be achieved in a single administrationprotocol.

Agents useful in the protocols of the present invention areidentifiable, for example, in accordance with the following procedures.A potential agent for free agent (i.e., non-targeted) administrationexhibits one or more of the following characteristics:

(i) retains an expanded luminal volume following angioplasty (e.g.,PTCA, percutaneous transluminal angioplasty (PTA) or the like) or othertrauma, including atheroectomy (e.g., rotoblater, laser and the like),coronary artery bypass procedures or the like; or resulting fromvascular disease (e.g., atherosclerosis, eye diseases secondary tovascular stenosis or atrophy, cerebral vascular stenotic diseases or thelike);

(ii) the initial increase in luminal area facilitated by the agent doesnot result in or accentuate chronic stenosis of the lumen;

(iii) inhibits target cell contraction or migration; and

(iv) is cytostatic.

Preferably, a therapeutic agent employed herein will have all fourproperties; however, the first and third are more important than thesecond and fourth for practice of the present invention. Cytochalasin B,for example, was evaluated to determine suitability for use in freetherapeutic agent protocols. The biological stenting effect ofcytochalasin B is achievable using a single infusion of the therapeuticagent into the traumatized region of the vessel wall at a doseconcentration ranging from about 0.1 microgram/ml to about 1.0micrograms/ml.

An agent useful in the sustained release embodiments of the presentinvention exhibits one or more of the following characteristics:

(i) retains an expanded luminal volume following angioplasty (e.g.,PTCA, percutaneous transluminal angioplasty (PTA) or the like) or othertrauma, including atheroectomy (e.g., rotoblater, laser and the like),coronary artery bypass procedures or the like; or resulting fromvascular disease (e.g., atherosclerosis, eye diseases secondary tovascular stenosis or atrophy, cerebral vascular stenotic diseases or thelike);

(ii) inhibits target cell proliferation (e.g., following 5 minute and 24hour exposure to the agent, in vitro vascular smooth muscle tissuecultures demonstrate a level of inhibition of ³H-thymidine uptake and,preferably, display relatively less inhibition of ³H-leucine uptake);

(iii) at a dose sufficient to inhibit DNA synthesis, produces only mildto moderate

(e.g., grade 2 or 3 in the assays described below) morphologicalcytotoxic effects;

(iv) inhibits target cell contraction; and

(v) is cytostatic.

Upon identification of a therapeutic agent exhibiting one or more of thepreceding attributes, the agent is subjected to a second testingprotocol that involves longer exposure of vascular smooth muscle cellsto the therapeutic agent.

An agent useful in the sustained release embodiments of the presentinvention exhibits the following characteristics:

(i) upon long term (e.g., 5 days) exposure, the agent produces the sameor similar in vitro effect on vascular smooth muscle tissue culture DNAsynthesis and protein synthesis, as described above for the 5 minute and24 hour exposures; and

(ii) at an effective dose in the long term in vitro assay for DNAsynthesis inhibition, the agent exhibits mild to moderate morphologicalcytotoxic effects over a longer term (e.g., 10 days).

Further evaluation of potential anti-proliferative agents within thepresent invention is conducted in an in vivo balloon traumatized pigfemoral artery model. Preferably, such agents demonstrate a 50% orgreater inhibition of cell proliferation in the tunica media vascularsmooth muscle cells, as indicated by a 1 hour “BRDU flash labeling”prior to tissue collection and histological evaluation. If an agent iseffective for a period of time sufficient to inhibit intimal smoothmuscle proliferation 50% or greater with a single exposure, it is anagent within the present invention that does not require administrationin a sustained release dosage form. Agents having shorter durationactivity are evaluated for sustained release if the systemic toxicityand potential therapeutic index appear to permit intravenousadministration to achieve the 50% inhibition, or if the agent isamenable to local delivery to the vascular smooth muscle cells withsustained release at an effective anti-proliferative dose. Sustainedrelease agents are evaluated in a sustained release dosage form for doseoptimization and efficacy studies. Preferably, anti-proliferative agentsuseful in the practice of the present invention decrease vascularstenosis by 50% in balloon traumatized pig femoral arteries and, morepreferably, to decrease vascular stenosis to a similar extent in pigcoronary arteries. Such agents are then evaluable in human clinicaltrials.

Cell proliferation (i.e., DNA synthesis) inhibition is the primarycharacteristic for sustained release of agents. Staurosporin, forexample, exhibits a differential between ³H-leucine and ³H-thymidineuptake such that it is cytostatic at administered doses. Longer durationcytotoxicity studies did not indicate that prolonged exposure to thetherapeutic agent would adversely impact the target cells. In addition,BRDU pulsing indicated that staurosporin inhibits target cellproliferation. Any convenient method for evaluating the capability ofinhibiting cell proliferation may alternatively be employed, however.Consequently, staurosporin is effective in retaining an expanded luminalvolume.

High levels of lipoprotein Lp(a) are known to constitute a major riskfactor for atherosclerosis, coronary heart disease and stroke. Onesymptom associated with such conditions and other problems, such asrestenosis following balloon angioplasty and other pathogenicconditions, is the proliferation or the migration of smooth musclecells. No direct link between Lp(a) and proliferation of vascular smoothmuscle cells had been established in the prior art.

An in vivo pathway for the modulation of vascular smooth muscle cellproliferation is shown in FIG. 15. This mechanism is believed toconstitute a portion of the mechanism that maintains vascular smoothmuscle cells in a non-proliferative state in healthy vessels. Thepathway has been elucidated by the inventors of a patent applicationfiled on even date herewith, entitled Prevention and Treatment ofPathologies Associated with Abnormally Proliferative Smooth MuscleCells.

Vascular smooth muscle cell proliferation is inhibited by an active formof TGF-beta. Tamoxifen has been shown by the experimentation detailed inExample 16 hereof to stimulate both the production and the activation ofTGF-beta. Heparin stimulates the activation of TGF-beta by affecting therelease of the active form of TGF-beta from inactive complexes presentin serum. TGF-beta neutralizing antibodies inhibit the activity ofTGF-beta, thereby facilitating the proliferation of vascular smoothmuscle cells. The apparent in vivo physiological regulator of theactivation of TGF-beta is plasmin. Plasmin is derived from plasminogenthrough activation by, for example, tPA (tissue plasminogen activator).Plasminogen and, therefore, plasmin activity is inhibited by thelipoprotein Lp(a) or apolipoprotein(a) (apo(a)), thereby decreasing theactivation of the latent form of TGF-beta and facilitating proliferationof vascular smooth muscle cells.

An additional pathway for the modulation of vascular smooth muscle cellproliferation is shown in FIG. 16. Resting smooth muscle cellsconstitute cells in their normal, quiescent non-proliferative state.Such resting smooth muscle cells may be converted to proliferatingsmooth muscle cells through activation by platelet derived growth factor(PDGF), fibroblast growth factor (FGF) or other stimulatory moieties.The proliferating smooth muscle cells may be converted to continualproliferating smooth muscle cells (i.e., smooth muscle cells capable ofgenerating a pathological state resulting from over-proliferationthereof) by an autocrine growth factor. This growth factor is believedto be produced by proliferating smooth muscle cells. An increased levelof autocrine growth factor, which can be inhibited by the active form ofTGF-beta or an appropriately structured (e.g., designed) small moleculeinhibitor, is believed to mediate the production of continualproliferating smooth muscle cells.

Lp(a) consists of low density lipoprotein (LDL) and apo(a). Apo(a)shares approximately 80% amino acid identity with plasminogen (seeMacLean et al., Nature, 330:132, 1987). Lp(a) has been found to inhibitcell-associated plasminogen activity (see, for example, Harpel et al.,Proc. Natl. Acad. Sci. USA, 86:3847, 1989). Experiments conducted onhuman aortic vascular smooth muscle cells derived from healthytransplant donor tissue, cultured in Dulbecco's modified Eagles medium(DMEM) +10% fetal calf serum (FCS) as described in Grainger et al.,Biochem. J., 283:403, 1992, indicated the following:

1) Addition of Lp(a) to sub-confluent human vascular smooth muscle cellsstimulated their proliferation in a dose dependent manner (addition of500 nM Lp(a) to human vascular smooth muscle cells caused a reduction indoubling time from 82+/−4 hours to 47+/−4 hours);

2) Addition of apo(a) had a similar effect, although a higherconcentration of apo(a) appeared to be required therefor; and

3) Addition of LDL at varying concentrations up to 1 micromolar had noeffect on proliferation.

One possible mode of action for Lp(a) and apo(a) is competitiveinhibition of surface-associated plasminogen activation and thesubsequent activation of TGF-beta by plasmin. TGF-beta is a potentgrowth inhibitor of a number of anchorage-dependent cells, includingsmooth muscle cells. TGF-beta is produced as a latent propeptide havinga covalently linked homodimer structure in which the active moiety isnon-covalently linked to the amino-terminal portion of the propeptide.Latent TGF-beta must be cleaved (e.g., in vitro by acid treatment or invivo by the serine protease plasmin) in order to become capable ofinhibiting the proliferation of vascular smooth muscle cells. Plasmin istherefore a leading candidate to be a physiological regulator ofTGF-beta.

The hypothesis that Lp(a) and apo(a) were acting on cultured humanvascular smooth muscle cells by interfering with activation of latentTGF-beta was tested. In support of this hypothesis, an observation wasmade that plasmin activity associated with vascular smooth muscle cellswas reduced 7-fold by Lp(a) and 5-fold by apo(a). The plasmin activityin the conditioned medium was also reduced by Lp(a) and apo(a) by about2-fold, but was much lower than cell-associated plasmin activity invascular smooth muscle cell cultures. These observations are consistentwith previous findings that Lp(a) is a more potent inhibitor ofsurface-associated, rather than fluid phase, plasminogen activation.

To exclude the possibility that Lp(a) was affecting the synthesis ofplasminogen activators rather than plasminogen activation, plasminogenactivator levels in human vascular smooth muscle cell cultures weremeasured in the presence and absence of the lipoproteins and in thepresence of a large excess of plasminogen, so that the lipoproteinspresent would not significantly act as competitive inhibitors. Totalplasminogen activator activity was not affected by the presence of anyof the lipoproteins in the vascular smooth muscle cell cultures. Forexample, plasminogen activator activity in the conditioned mediumremained at 0.7+/−0.06 mU/ml with Lp(a) additions up to 500 nM.

Lp(a) and apo(a) both reduced the level of active TGF-beta by more than100-fold compared to control or LDL-treated cultures. The level of totallatent plus active TGF-beta measured by ELISA as described in Example 16was unaffected by the presence of Lp(a) or apo(a), however. These factslead to the conclusion that Lp(a) stimulates proliferation of humanvascular smooth muscle cells by inhibiting plasmin activation of latentTGF-beta to active TGF-beta.

To further test this conclusion and exclude the possibility that Lp(a)was acting by binding active TGF-beta as well as reducing plasminactivity, human vascular smooth muscle cells were cultured in thepresence of Lp(a). These cells had a population doubling time of 47+/−3hours. Addition of plasmin was able to overcome the population doublingtime reducing effect of Lp(a) and reduce the cell number to controllevels, with the population doubling time increased to 97+/−4 hours.

The role of plasmin in the pathway was confirmed by studies in whichinhibitors of plasmin activity were added to human vascular smoothmuscle cells. Like Lp(a), these protease inhibitors increased cellnumber. Aprotinin, for example, decreased the population doubling timefrom 82+/−4 hours in control cultures to 48+/−5 hours, andalpha2-antiplasmin decreased the population doubling time to 45+/−2hours. 500 nM Lp(a) and aprotinin addition resulted in only a slightadditional stimulation of proliferation, with the population doublingtime for cultures of this experiment being 45+/−6 hours. Neutralizingantibodies to TGF-beta similarly decreased population doubling time invascular smooth muscle cells (see, for example, Example 16). In summary,Lp(a), plasmin inhibitors and neutralizing antibody to TGF-betastimulate proliferation of vascular smooth muscle cells, while plasminnullifies the growth stimulation of Lp(a). These results support thetheory that the mode of action of Lp(a) and apo(a) is the competitiveinhibition of plasminogen activation.

Experimentation conducted to ascertain the impact of tamoxifen onTGF-beta and vascular smooth muscle cell proliferation is set forth indetail in Example 16. The results of those experiments are summarizedbelow.

1) Addition of tamoxifen decreased the rate of proliferation, withmaximal inhibition observed at concentrations above 33 micromolar. 50micromolar tamoxifen concentrations produced an increase in cell number(96 hours following the addition of serum) that was reduced by66%+/−5.2% (n=3).

2) Tamoxifen did not significantly reduce the proportion of cellscompleting the cell cycle and dividing. Inhibition of vascular smoothmuscle cells caused by tamoxifen therefore appears to be the result ofan increase in the cell cycle time of nearly all (>90%) of theproliferating cells.

3) Tamoxifen decreases the rate of proliferation of serum-stimulatedvascular smooth muscle cells by increasing the time taken to traversethe G₂ to M phase of the cell cycle.

4) Tamoxifen decreased the rate of proliferation of vascular smoothmuscle cells by inducing TGF-beta activity.

5) Vascular smooth muscle cells produced TGF-beta in response totamoxifen. Tamoxifen appears to increase TGF-beta activity in culturesof rat vascular smooth muscle cells by stimulating the production oflatent TGF-beta and increasing the proportion of the total TGF-betawhich has been activated.

6) Tamoxifen, unlike heparin, does not act by releasing TGF-beta frominactive complexes present in serum.

7) TGF-betal mRNA was increased by approximately 10-fold by 24 hoursafter addition of tamoxifen (10 micromolar). This result suggests thatthe expression of TGF-beta mRNA by the smooth muscle cells will beincreased, thereby facilitating decreased proliferation thereof byactivated TGF-beta. This mechanism can be exploited using cellsincorporating nucleic acids encoding TGF-beta mRNA, which cells areidentifiable by persons skilled in the art employing known techniques.

8) Tamoxifen is a selective inhibitor of vascular smooth muscleproliferation with an ED₅₀ at least 10-fold lower for vascular smoothmuscle cells than for adventitial fibroblasts.

Additional experimentation has shown that the addition of Lp(a) orapo(a) substantially reduced the vascular smooth muscle cellproliferation inhibitory activity of tamoxifen, with the populationdoubling time in the presence of tamoxifen and Lp(a) being 42+/−2 hours.Also, the presence of Lp(a) reduced the levels of active TGF-betaproduced in response to the addition of tamoxifen by about 50-fold.Addition of plasmin to rat vascular smooth muscle cells treated withtamoxifen and Lp(a) resulted in most of the TGF-beta being activated,and proliferation was again slowed (with the population doubling timebeing 57+/−3 hours). These observations are consistent with the theorythat Lp(a) acts by inhibiting TGF-beta activation.

Identification of therapeutic agents (direct or indirect TGF-betaactivators or production stimulators) that act to inhibit vascularsmooth muscle cell proliferation by the pathway shown in FIG. 15 can beidentified by a practitioner in the art by conducting experiments of thetype described above and in Example 16. Such experimental protocolsfacilitate the identification of therapeutic agents useful in thepractice of the present invention and capable of one of the followingactivities:

1) activation or production of TGF-beta;

2) having TGF-beta activity;

3) activation of plasmin;

4) activation of plasminogen; and

5) reduction of Lp(a) or apo(a) level.

Having TGF-beta activity includes, but is not limited to, disruption ofcyclin-dependent protein kinase (CDK) transformation from a slowmigrating form to a rapid migrating form, disruption of CDK-cyclincomplex formation or activation or the like.

Identification of therapeutic agents (direct or indirect TGF-betaactivators or production stimulators) that act to inhibit vascularsmooth muscle cell proliferation by the pathway shown in FIG. 16 can beidentified by a practitioner in the art by conducting experimentationusing known techniques that is designed to identify growth factors madeby proliferating smooth muscle cells, pericytes, lymphorecticular cellsor the like, which growth factors also act on those cells (i.e.,autocrine growth factors). Known techniques for rational drug design arethen used to screen small molecules for the ability to inhibit theproduction or activity of such autocrine growth factors. Suchexperimental protocols facilitate the identification of therapeuticagents useful in the practice of the present invention and capable ofone of the following activities:

1) production or activation of TGF-beta;

2) having TGF-beta activity; and

3) inhibit the activity or production of an autocrine growth factorproduced by proliferating smooth muscle cells.

Smooth muscle cell proliferation is a pathological factor in myocardialinfarctions, atherosclerosis, thrombosis, restenosis and the like.Therapeutic agents of the present invention, including tamoxifen,TGF-beta and the like, having at least one of the activities recitedabove and therefore being capable of inhibiting proliferation ofvascular smooth muscle cells, are useful in the prevention or treatmentof these conditions. Manipulation of the proliferation modulationpathway for vascular smooth muscle cells to prevent or reduce suchproliferation removes or reduces a major component of the arteriallesions of atherosclerosis and the restenosed arteries followingangioplasty, for example.

More specifically, chronically maintaining an elevated level ofactivated TGF-beta reduces the probability of atherosclerotic lesionsforming as a result of vascular smooth muscle cell proliferation.Consequently, administration of TGF-beta, TGF-beta activators orTGF-beta production stimulators protects against atherosclerosis andsubsequent myocardial infarctions that are consequent to coronary arteryblockage. Also, substantially increasing the activated TGF-beta levelfor a short time period allows a recipient to at least partially offsetthe strong stimulus for vascular smooth muscle cell proliferation causedby highly traumatic injuries or procedures such as angioplasty.Continued lower dose delivery to the traumatized site further protectsagainst restenosis resulting from vascular smooth muscle cellproliferation in the traumatized area.

Other embodiments of the present invention involve the administration oftaxol or analogs thereof in soluble or sustained release dosage form.Taxol is believed to stabilize vascular smooth muscle cells againstdivision by binding to microtubules and inhibiting the organization andordering of the microtubule network. Cell migration may also beinhibited by this mechanism. Taxotere, an exemplary taxol analog, has adifferent method of action, but also inhibits cell division.

The invention will be better understood by making reference to thefollowing specific examples.

Example 1 Binding to Vascular Smooth Muscle Cells in the Blood VesselWall In Vivo

FIG. 1B illustrates the binding of NR-AN-01 (a murine IgG2b MAb) to thesmooth muscle cells in the vascular wall of an artery in a 24-year oldmale patient, 4 days after the i.v. administration of NR-AN-01. FIG. 1Bis a photomicrograph of a histological section taken through the medialregion of an arterial wall of the patient after NR-AN-01 administration,where the section was reacted ex vivo with HRP-conjugated goatanti-mouse IgG. The reaction of the HRP-conjugate with NR-AN-01 MAb wasvisualized by adding 4-chloro-1-naphthol or 3,3′-diaminobenzidinetetrahydrochloride as a peroxidase substrate (chromogen). The reactionproduct of the substrate forms an insoluble purple or dark brownprecipitate at the reaction site (shown at #2, FIG. 1B). A counter stainwas used to visualize collagenous extracellular matrix material (shownat #2, FIG. 1B) or cell nuclei (#1, FIG. 1B). Smooth muscle cells arevisualized under microscopic examination as purple stained cells (FIG.1A and FIG. 1B). This photomicrograph (FIG. 1B) demonstrates the abilityof the MAb to specifically bind to human vascular smooth muscle in vivo,and to be internalized by the cells and remain in the cells for extendedperiods.

Example 2 Therapeutic Conjugates Containing Trichothecene TherapeuticAgents

Conjugates of NR-AN-01 and Roridin A were constructed by chemicallycoupling a hemisuccinate derivative of the trichothecene cytotoxin (asdescribed below) to a monoclonal antibody designated NR-AN-01. Twoconjugates were prepared, one coupled at the Roridin A 2′ position andone at the 13′ position. Two schemes were used in this synthesis, asdepicted in FIG. 2 and FIG. 3. The conjugate was then purified fromunreacted Roridin A by PD-10 SEPHAROSE® column chromatography(Pharmacia; Piscataway, N.J.), analyzed by size exclusion high pressureliquid chromatography, and the column fractions were characterized bySDS-PAGE and isoelectric focusing (IEF), as described below.

FIG. 2 shows diagrammatically the first reaction scheme for synthesis ofRoridin A hemisuccinyl succinimidate (RA-HS-NHS) through a two stepprocess with reagents: succinic anhydride, triethylamine (NEt₃) anddimethyl amino pyridine (DMAP) present in dichloromethane (CH₂Cl₂) atroom temperature (RT); and, N-hydroxysuccinimide (NHS) and dicyclohexylcarbodiimide (DCC) reagents also in CH₂Cl₂ at RT.

FIG. 3 shows diagrammatically the second reaction scheme for synthesisof Roridin A hemisuccinyl succinimidate (RA-HS-NHS) through a five stepprocess with reagents: t-butyl dimethyl silyl chloride (TBMS-Cl) andimidazole in dimethylformamide (DMF) at room temperature (RT); aceticanhydride, triethylamine (TEA), and diethylaminopyridine indichloromethane (CH₂Cl₂) at RT; succinic anhydride, triethylamine (TEA)and dimethylaminopyridine (DMAP) in (CH₂Cl₂) at RT; and,N-hydroxysuccinimide (HS) and dicyclohexyl carbodiimide (DCC) reagents.

Synthesis of 2′ Roridin-A Hemisuccinic Acid (2):

To 0.5 g (0.94 mmol) of Roridin A, 15 ml of dichloromethane was added.To this solution with stirring was added 0.104 g (1.04 mmol) of succinicanhydride. To the reaction mixture, 0.2 ml of triethylamine in 5 mldichloromethane was added. To the homogeneous reaction mixture, acatalytic amount of dimethylaminopyridine was added and stirred at roomtemperature for 15 hours. Completion of the reaction was followed bythin layer chromatography CH₂Cl₂: CH₃OH=9.7:0.3 with few drops of aceticacid). At the end of the reaction, 0.3 ml of glacial acetic acid wasadded and the solvent removed under reduced pressure. The dried cruderesidue was partitioned between water and methylene chloride. Thecombined methylene chloride extracts (3×50 ml) were dried over anhydroussodium sulfate, solvent was removed under vacuum and dried to yield0.575 g (96%) of a crude mixture of three compounds. Preparative C18HPLC separation of the crude mixture in 50% acetonitrile-water with 2%acetic acid yielded 0.36 g (60%) of 2 as a white solid.

Synthesis of Succinimidyl 2′-Roridin A Hemisuccinate (3):

To 0.3 g (0.476 mmol) of 2′ Roridin A hemisuccinic acid in 30 mldichloromethane, 0.055 g (0.478 mmol) N-hydroxysuccinimide was added. Tothe clear reaction mixture, 0.108 g (0.524 mmol)dicyclohexylcarbodiimide was added. The reaction mixture was stirred atroom temperature for 6 hours. Completion of the reaction was followed byTLC CH₂Cl₂: CH₃OH=9.7:0.3 with a few drops of acetic acid) as adeveloping solvent. A few drops of glacial acetic acid was added to thereaction mixture and the solvent was removed under reduced pressure. Tothe dried residue dichloromethane was added and the precipitated DCU wasfiltered. Solvent from the filtrate was removed under reduced pressureto yield a white solid. From the crude product, 0.208 g (60%) of 3 waspurified by preparative HPLC in 50% acetonitrile with 2% acetic acid asa mobile phase.

Synthesis of 13′-t-Butyldimethylsilyl Roridin A (4):

To 72.3 mg (0.136 mmol) of Roridin A in 0.5 ml dimethylformamidesolution, 0.055 g (0.367 mmol) t-butyldimethylsilyl chloride and 0.025 g(0.368 mmol) of imidazole were added. The reaction mixture was stirredat room temperature for 15 hours. Completion of the reaction wasfollowed by silica gel thin layer chromatography using 1% MeOH—CHCl₃ asa developing solvent. Solvent from the reaction mixture was removed invacuo and dried. The crude product was partitioned between water andmethylene chloride. Solvent from the combined methylene chlorideextracts was removed under reduced pressure and dried. The crude productwas purified by flash chromatography using EtOAc:Hexane (1:3) as aneluting solvent. Solvent from the eluants was removed under reducedpressure to yield 0.66 g (75%) of 4 as a solid.

Synthesis of 13′-t-Butyldimethylsilyl 2′ Acetyl Roridin A (5):

To 0.1 g (0.155 mmol) of 13′-t-butyldimethylsilyl Roridin A in 10 mldichloromethane, 0.3 ml acetic anhydride, 0.2 ml triethylamine and a fewcrystals of dimethylaminopyridine were added and stored at roomtemperature for 2 hours. Completion of the reaction was followed by TLCin 1% methanol-methylene chloride as a developing solvent. Solvent wasremoved under reduced pressure and purified by a silica gel column using1% methanol-chloroform as an elution solvent. Solvent from the eluantswas removed under vacuum to yield 0.085 g (80%) of 5 as a solid.

Synthesis of 2′ Acetyl Roridin A (6):

To 0.05 g (0.073 mmol) of 2′ acetyl 13′-t-butyldimethylsilyl Roridin Ain 5 ml tetrahydrofuran, 0.3 ml of 1 M tetrabutyl-ammonium fluoridesolution in THF was added. The reaction mixture was stirred at roomtemperature for 2 hours. Completion of the reaction was followed bysilica gel thin layer chromatography using 1% MeOH—CHCl₃ as thedeveloping solvent. Solvent from the reaction mixture was removed underreduced pressure and dried. The crude product was purified on a silicagel column using 1% CH₃OH—CHCl₃ as an eluting solvent. Solvent from thecombined eluants were removed under vacuum to yield 0.020 g (48%) of 6as a solid.

Synthesis of 2′-Acetyl 13′-Hemisuccinyl Roridin A (7):

To 0.05 g (0.087 mmol) of 2′-acetyl Roridin A in 1 ml ofdichloromethane, 0.025 g (0.25 mmol) succinic anhydride and 35 ml oftriethylamine was added. A few crystals of dimethylaminopyridine wasadded as a catalyst. The reaction mixture was stirred at roomtemperature for 24 hours. Completion of the reaction was followed bythin layer chromatography using 5% MeOH—CH₂Cl₂ as developing solvent. Atthe end of the reaction 30 ml of glacial acetic acid was added. Solventfrom the reaction mixture was removed under reduced pressure and dried.The crude product was partitioned between water and ethyl acetate.Solvent from the combined ethyl acetate fractions was removed underreduced pressure. Crude product was purified by passing through a silicagel column to yield 0.039 g (66%) of 7 as a white solid.

Synthesis of Succinimidyl 2′-Acetyl 13′-Roridin A Hemisuccinate (8):

To 0.036 g (0.0050 mmol) of 2′-acetyl 13′-Roridin A hemisuccinic acid in2 ml dichloromethane, 0.009 g (0.09 mmol) N-hydroxysuccinimide wasadded. To a stirred solution, 0.012 g (0.059 mmol)dicyclohexylcarbodiimide was added. The reaction mixture was stirred atroom temperature for 8 hours. Completion of the reaction was followed bysilica gel thin layer chromatography using 5% MeOH—CH₂Cl₂ as adeveloping solvent. A few drops of glacial acetic acid was added to thereaction mixture. Solvent from the reaction mixture was removed underreduced pressure and dried. The crude product was purified on a silicagel column using 5% MeOH—CH₂Cl.₂ as an eluting solvent. Solvent from thecombined eluants was removed under vacuum to yield 0.025 g (61%) of 8 asa white solid.

Conjugation of Succinimidyl 2′-Roridin A Hemisuccinate (3) andSuccinimidyl 2′-Acetyl 13′-Roridin A Hemisuccinate (8) to NR-AN-01 WholeAntibody (MAb):

Conjugation reactions were performed at pH 8.0 in borate buffer in thepresence of 25% dimethylsulfoxide (DMSO) solvent at room temperaturewith gentle mixing for 45 minutes prior to purification by gelpermeation chromatography. The molar trichothecene drug precursor toantibody offerings were 25:1 and 40:1 for the 2′ and 13′ Roridin Aanalogues (3 and 8), respectively. Antibody concentration was 0.9 to 1.0mg/ml during the conjugation reaction.

A Typical 2′ Analogue (3) Reaction with 25 mg of Antibody was asfollows:

To 4.7 ml of 5.3 mg Ab/ml in phosphate buffered saline (i.e., PBS; 150mM NaCl, 6.7 mM Phosphate, pH 7.3) was added 10 ml PBS and 5 ml ofborate buffer (0.5 M, pH 8.0). With stirring gently to the reactionmixture, 6.3 ml of DMSO containing 1.37 mg of succinimidyl 2′ Roridin Ahemisuccinate (3) was then added dropwise over a 15 second period.

Purification:

To purify, one ml reaction aliquots were applied to Pharmacia PD-10Sepharose<columns equilibrated in PBS. The eluted conjugate wascollected in 2.4 to 4.8 ml fractions. The PD-10 purified conjugatealiquots were then pooled and concentrated on an Amicon PM-10 DiAflo®concentrator to 1.5 to 2.0 mg of Ab/ml; sterile filtered through a 0.2μGelman Acrodisc® and filled into sterile glass vials in 5 ml volume.

The 2′ conjugate was quick frozen in liquid nitrogen and then stored at−70° C. until use. The 13′ Roridin A NR-AN-01 conjugate was storedfrozen or refrigerated (i.e., 5-10° C.).

Characterization of Conjugates:

Protein concentration was determined by BCA assay using the copperreagent method (Pierce Chemical Corp.).

Assessment of degree of antibody derivatization was performed by firsthydrolyzing an aliquot of conjugate in 0.2 M carbonate, pH 10.3 for 4hours (at room temperature for 2′ conjugate or at 37° C. for the 13′conjugate) followed by filtration through a PM-30 membrane. The filtratewas then assayed for Roridin A on C-18 reverse phase HPLC using a mobilephase of 50:48:2 ratio CH₃CN:H₂O:HOAC, respectively. A 1.32 correctionfactor was used to correct for parallel macrocyclic ring decompositionthat gives polar products during the hydrolysis of the 13′ conjugate.

Size exclusion chromatography on DuPont Zorbax® HPLC and isoelectricfocusing Serva® gel plates (pH 3 to 10) were also performed. Noindication of aggregation was observed by HPLC.

Immunoassay of the Roridin A-antibody conjugates was performed by eithercompetitive ELISA using biotinylated-Ab with Streptavidin/Peroxidasedetection or by a competitive cell binding assay using ¹²⁵I-labeledantibody. Alternatively, immunoreactivity was measured under conditionsof antigen saturation in a cell binding assay wherein antibody was firsttrace labeled with I-125 by the chloramine T method and thensubsequently derivatized with 2′ and 13′ Roridin A precursors.

The structural formula of the trichothecene is shown below:

Example 3 Kinetics of Binding to Smooth Muscle Cells

For administration by i.v. catheter, it is desirable that thetherapeutic conjugates of the invention be administered in less than 3to 5 minutes, so that blood flow can be reestablished in the patient.Therefore, studies were conducted to determine the binding kinetics of asmooth muscle binding protein with a Ka of >10⁹ liter/mole. Becausehuman vascular smooth muscle cells grow slowly in culture, and baboonsmooth muscle cells were found to express the human CSPG cell surfacemarker, BO54 baboon artery smooth muscle cells and human A375 M/M(melanoma; ATCC #CRL1619) cells bearing CSPG surface marker were used inmany of the studies described in the Examples, below.

For the kinetic binding studies, A375 M/M and BO54 cells were seeded insterile 96 well microtiter plates at 2500 cells/well. Plates werewrapped in aluminum foil, and incubated at 37° C. overnight in ahumidified atmosphere of 5% CO₂/95% air. After approximately 18 hr,incubation plates were removed and cells were fixed with 0.05%glutaraldehyde for 5 minutes to prevent membrane turnover. Followingfixation, the plates were exhaustively washed with PBS containing 0.5%Tween-20®. Serial two-fold dilutions of an NR-AN-01 therapeuticconjugate containing Roridin A were prepared at protein concentrationsof 10 mg/ml to 20 ng/ml, and each dilution was aliquoted into two wells.The plates were incubated at 4° C. with the NR-AN-01 for 5, 15, 30, and60 minutes, after which the unbound protein was removed by aspirationand 100 ml of CS buffer was added (5% chicken serum/0.5% Tween-20® inPBS) to each well. CS buffer was removed and the NR-AN-01 therapeuticconjugate bound to the cells was visualized by adding 100 ml ofHRP-conjugated goat anti-mouse IgG (Sigma Chemical Co., St. Louis, Mo.)to each well; incubating at 4° C. for 1 hr.; washing with PBS/0.05%Tween® to remove unbound goat IgG; and, adding2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid (ABTS) chromogenicsubstrate (i.e., for HRP). After incubating for 30 minutes, the amountof NR-AN-01 bound to the cells was quantified by measuring theabsorbance at 415 nm and 490 nm using an ELISA plate reader equipped fordata acquisition by a Compaq computer.

FIG. 4A graphically depicts the results of in vitro studies in whichA375 m/m marker-positive cells were held at 4° C. (i.e., to preventmembrane turnover) for 5 minutes (open squares, FIG. 4A), 15 minutes(closed diamonds, FIG. 4A), 30 minutes (closed squares, FIG. 4A) or 60minutes (open diamonds, FIG. 4A) with different concentrations ofNR-AN-01 (NRAN01 μg/ml). The binding of the NR-AN-01 MAb to the A375cells was quantified by washing to remove unbound antibody, addingHRP-conjugated goat anti-mouse IgG to react with the cell-bound MAb,washing to remove unbound goat second antibody, and adding2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) substratefor peroxidase. Color development was monitored after 30 minutes at both415 nm and 490 nm (ABS415, 490).

FIG. 4B graphically depicts the results of in vitro studies conducted ina manner similar to those described above in regard to FIG. 4A, butusing BO54 marker-positive smooth muscle cells, i.e., instead of theA375 m/m cells.

The results presented in FIG. 4A and FIG. 4B show significant binding ofNR-AN-01 to A375 and BO54 cells within 5 minutes at 4° C., even at thelowest dose of 20 ng/ml.

Example 4 Effects of Roridin A and RA-NR-AN-01 Conjugates

The effects of Roridin A (RA) and RA-NR-AN-01 conjugates on cellularprotein synthesis (i.e., by ³H-leucine incorporation) and metabolicactivity (i.e., by mitochondrial MTT assay) were tested in theexperiments detailed in EXAMPLE 5 and EXAMPLE 6, below. The studies inEXAMPLE 4 include experiments to determine the effects of long-term(i.e., 24 hour) treatment with the agents. The studies in EXAMPLE 5include experiments to determine the effects of “pulse” (i.e., 5 minute)treatment on cells. In both studies, the cellular specificity of theeffects were evaluated by including “target” cells (i.e., cells bearingthe CSPG “marker”) and non-target cells. For comparative purposes,free-RA (i.e., uncoupled) was also included in the studies. The effectson cellular protein synthesis or metabolic activity were evaluatedeither immediately following the treatment, or a “recovery period” wasallowed (i.e., involving incubation of the cells overnight at 37° C.) todetermine the long-term effects of the agents on the cell populations.

Metabolic Effects After 24 Hours Exposure:

While it is known that monoclonal antibody-drug conjugates may have adegree of specificity for cells bearing marker antigens when employed invivo, it has proven more difficult in many systems to demonstrate invitro specificity of action, especially with compounds that arelipophilic. Therefore, the present experiments were conducted in whichthe inhibitory effects of the NR-AN-01-Roridin A conjugate was tested ontarget and non-target cells over 24 hours. The results with RA-NR-AN-01were compared to the effect of free Roridin A over the same 24-hourperiod. A modified methyl-tetrazolium blue (MTT) assay was utilized with3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (Sigma) todetermine cellular metabolic activity. This assay is thought to measurecellular mitochondrial dehydrogenase activity. For some of thesestudies, M14 (melanoma) and BO54 (smooth muscle) cell lines were used asmarker-positive target cells and HT29 cells (colon carcinoma; ATCC#HTB38) were used as the non-target specificity control. In otherstudies, A375 was used as a marker-positive cell. The HT29 and M14 cellswere seeded in 96-well microtiter plates at a concentration of 5.0×10³cells/well, and the BO54 cells were seeded at 2.5×10³ cells/well. Serialtwo-fold dilutions of free Roridin A and 2′RA-HS-NR-AN-01 (i.e., RoridinA coupled through a hemisuccinate (HS) coupling agent at the 2′ positionto NR-AN-01) were prepared in DMEM over a range of proteinconcentrations from 20 mg/ml to 40 pg/ml. Test agents were added (induplicate) to microtiter wells (100 ml/well), and the plates werewrapped in aluminum foil and incubated at 37° C. in a humidifiedatmosphere consisting of 5% CO₂/95% air for 24 hours. After 24 hours,medium was removed (by aspiration), fresh DMEM was added (100 ml/well),and the cells were returned to incubate for an additional overnight(i.e., 16-18 hours) “recovery period”. At the end of the “recoveryperiod” cellular metabolic activity was determined by adding 20 ml toeach well of a 5 mg/ml MTT solution. The plates were covered andincubated at 37° C. for 4 hours and then the reaction was developed byadding 100 ml/well of 10% SDS/0.1 N HCl. The dark blue solubilizedformazan reaction product was developed at room temperature after 16-18hours and quantified using an ELISA microtiter plate reader at anabsorbance of 570 nm.

FIG. 5A graphically depicts the results of in vitro studies in whichBO54 marker-positive smooth muscle cells were incubated with differentconcentrations of RA-NR-AN-01 (NRAN01-RA; open squares, FIG. 5A) or freeRoridin A (Free RA; closed diamonds, FIG. 5A) for a period of 24 hours,washed, and then returned to culture for an additional 16-18 hourovernight (o/n) recovery period prior to testing metabolic activity inan MTT assay. The concentrations of Free RA and RA-NR-AN-01 areexpressed as the calculated concentration of Roridin A (in mg/ml plottedon a log scale) in the assay (i.e., rather than the total mg/ml ofNR-AN-01 protein in the assay), so that direct comparisons could bemade. The metabolic activity of the cells in the MTT assay is presentedas the percentage of the metabolic activity measured in a controluntreated culture of cells (i.e., % control).

FIG. 5B graphically depicts the results of in vitro studies conducted ina manner similar to those described above in regard to FIG. 5A, butcomparing the effects of only RA-NR-AN-01 (NRAN01-RA) on three differentcell types: namely, BO54 marker-positive smooth muscle cells(BO54-NRAN01-RA; open squares, FIG. 5B); HT29 marker-negative controlcells (HT29-NRAN01-RA; closed diamonds, FIG. 5B); and, M14marker-positive cells (M14-NRAN01-RA; closed squares, FIG. 5B). Asdescribed above in regard to FIG. 5A, the concentrations in the presentexperiment are expressed in terms of ug/ml of Roridin A. Metabolicactivity of the cells is expressed in a manner similar to that in FIG.5A, i.e., as the percentage of activity measured in an untreated controlculture of cells (% control).

The results presented in FIG. 5A and FIG. 5B show that metabolicactivity measured in the MTT assay was significantly decreased in allpopulations of test cells, even 16-18 hours after a 24-hour incubationin either free Roridin A or the 2′ or 13′ RA-NR-AN-01 conjugates. Theeffects of the RA-NR-AN-01-conjugates appeared to be non-specificallyinhibitory for both target (BO54 and M14) and non-target (HT29) cells(FIGS. 5A and 5B). The inhibitory effects were observed at a freeRoridin A or RA-conjugate concentration of >10 ng/ml.

For comparative purposes, a second study was conducted in which theeffects of Pseudomonas exotoxin (PE) conjugates on cells were evaluatedin a similar protocol. For these studies, target and non-target cellswere treated with PE or PE-NR-AN-01 for 24 hours, and then allowed a“recovery period” (as above) before metabolic activity was tested in anMTT assay.

FIG. 6A graphically depicts the results of in vitro studies conducted ina manner similar to those described above in regard to FIG. 5A, butdesigned to study the metabolic effects of PE-NR-AN-01 (NRAN01-PE) oncells, i.e., rather than RA-NR-AN-01. Three different cell types wereutilized: namely, BO54 marker-positive smooth muscle cells (BO54; opensquares, FIG. 6A); HT29 marker-negative control cells (HT29; closeddiamonds, FIG. 6A); and, M14 marker-positive cells (MT14; closedsquares, FIG. 6A). In this study, the concentration of conjugate isexpressed in μg/ml NR-AN-01 protein (plotted on a log scale), and themetabolic activity is expressed as the percentage of the MTT activitymeasured in an untreated control culture (% control).

FIG. 6B graphically depicts the results of in vitro studies conducted inmanner similar to those discussed above in regard to FIG. 6A, butdesigned to compare the effects obtained with free PE (PE) to thoseobtained above, i.e., in FIG. 6A, with PE-NR-AN-01. The cells, cultureconditions, calculations, and presentation of the results are the sameas in FIG. 6A, above.

The results presented in FIG. 6A and FIG. 6B show that 24 hours exposureto PE-NR-AN-01 or free PE was non-specifically inhibitory to cells atconcentrations of >100 ng/ml.

While this type of non-specific inhibition was judged to be of potentialvalue for biological atheroectomy, it was not considered desirable fortreatment of restenosis following angioplasty where dead and dying cellsmay release factors that stimulate smooth muscle proliferation.

Example 5 Effects of Pulse-Treatment on Cellular Activity

Additional studies were conducted to evaluate the effects of ashort-term, i.e., 5 minute, exposure to a Roridin A-containingtherapeutic conjugate on cells. In these studies, both metabolicactivity (measured in MTT assays) and cellular protein synthesis(measured by ³H-leucine incorporation) were evaluated.

Effects after 5 Minutes of Exposure: Protein Synthesis

The effects of a 5-minute exposure to free Roridin A (RA) or atherapeutic conjugate were evaluated. Roridin A-NR-AN-01 coupled througha hemisuccinyl (HS) at either the 2′ position (2′RA-HS-NR-AN-01) or the13′ position (13′RA-HS-NR-AN-01) were employed. (In the case of13′RA-HS-NR-AN-01, the 2′ position of Roridin A was also acetylated.)The RA, 2′ or 13′RA-NR-AN-01 conjugates were diluted two fold in sterileDMEM over a range of concentrations from 400 ng/ml to 780 pg/ml ofRoridin A. (The test samples were all normalized to Roridin A, so thatdirect comparisons could be made of the effects at comparable doses.)Samples were aliquoted (in duplicate) into duplicate microtiter platesat 100 ml/well and incubated at room temperature for five minutes.

Both short-term and long-term effects of the test samples onmarker-positive A375 and marker-negative HT29 cells were determined. Forstudying the short-term effects, 100 ml/well of [³H]-leucine (0.5mCi/ml) was added immediately after the 5-minute treatment withconjugate (or RA) and protein synthesis was evaluated over a four-hourperiod. For determining the long-term effects, the cells were treatedfor 5 minutes, washed, and then returned to culture for a 24-hour“recovery” period in DMEM medium containing either 5% NBS/5% Serum Plus®(i.e., for A375 or HT29 cells) or 10% FBS (i.e., for BO54 cells). At theend of the “recovery” period, the incubation medium was removed (i.e.,by aspiration) and ³H-leucine was added (as above). In both cases (i.e.,whether short-term or long-term), protein synthesis of the cells wasevaluated by incubating the cells with the 3H-leucine for 4 hours at 37°C. in a humidified chamber (as above), and all results are calculated bycomparison with non-treated cells (i.e., 100% control). After 4 hoursthe ³H-leucine was removed, the cells were removed from the substrata bytrypsin-treatment, aspirated (using a PHD™ cell harvester (CambridgeTechnology, Inc., Cambridge, Mass.)) and collected by filtration onglass fiber filters. The glass fiber filters were dried andradioactivity quantified by liquid scintillation spectroscopy in aBeckman liquid scintillation counter.

FIG. 7A graphically depicts the results of in vitro studies conducted toinvestigate the effects on control HT29 marker-negative cells of a 5minute exposure to different concentrations of Roridin A (Free RA; opensquares, FIG. 7A), or 2′RA-NR-AN-01 (2′RA-NRAN01; closed squares, FIG.7A), or 13′RA-NR-AN-01 (13′RA-NRAN01; closed triangles, FIG. 7A)conjugates. The concentrations of Free RA, 2′RA-NR-AN-01 or 13′NR-AN-01are expressed as the calculated concentration of Roridin A in the assay(in μg/ml plotted on a log scale), i.e., rather than the total μg/ml ofNR-AN-01 protein, so that direct comparisons of the results can be made.For these studies, the cells were treated for 5 minutes, washed, andthen returned to culture for 4 hours, during which time cellular proteinsynthesis was evaluated by adding 0.5 mCi/ml of ³H-leucine to theculture medium. At the end of the 4 hour period, cellular proteins werecollected and radioactivity was determined. The results are expressed asthe percentage of the radioactivity recorded in a control (non-treated)HT29 cell culture (i.e., % control).

FIG. 7B graphically depicts the results of in vitro studiesinvestigating the effects on control HT29 marker-negative cells of a 5minute expose to different concentrations of Free RA (open squares, FIG.7B), 2′RA-NRAN01 (closed squares, FIG. 7B), or 13′RA-NRAN01 (closedtriangles, FIG. 7B), as described above in regard to FIG. 7A, but in thepresent experiments the cells were incubated for a 16-18 hour recoveryperiod (i.e., overnight; o/n) prior to testing protein synthesis in afour hour ³H-leucine protein synthesis assay. The results are presentedin a manner similar to those above in FIG. 7A.

The results presented in FIG. 7A and FIG. 7B show the short-term andlong-term effects, respectively, of RA, 2′RA-HS-NR-AN-01, and13′RA-HS-NR-AN-01 on protein synthesis by HT29 control cells. Theresults show a dose-response inhibition of cellular protein synthesis bythe free Roridin A, but not by RA-NR-AN-01, in HT29 cells. Theinhibition triggered by RA during the 5 minutes of incubation was stillmanifest after the 16-18 hours recovery period (FIG. 7B). In contrast,treatment of non-target HT29 cells with 2′RA-HS-NR-AN-01 or13′RA-HS-NR-AN-01 did not result in detectable inhibition of proteinsynthesis. Thus, these results (in contrast to those obtained above over24 hours) seem to suggest a surprising degree of specificity to the invitro action of the NR-AN-01-conjugates when treatment was delivered ina 5-minute “pulse”. However, it was also possible that theNR-AN-01-conjugate was inactive, and so additional experiments wereconducted to evaluate the effect of the conjugates on target cells.

FIG. 7C graphically depicts the results of in vitro studiesinvestigating the effects on A375 m/m marker-positive cells of a 5minute exposure to different concentrations of Free RA (open squares,FIG. 7C), 2′RA-NR-AN-01 (closed squares, FIG. 7C) or 13′RA-NR-AN-01(closed triangles, FIG. 7C), as described above in regard to FIG. 7A. Inthe present studies, the A375 cells were incubated for 5 minutes in thetest agent, washed, and tested for protein synthesis over the next 4hours by adding 0.5 mCi/ml ³H-leucine to the culture medium. The resultsof the experiments are plotted in a manner similar to those described,above, in regard to FIG. 7A.

FIG. 7D graphically depicts the results of in vitro studiesinvestigating the effects on A375 m/ml marker-positive cells of a 5minute exposure to different concentrations of Free RA (open squares,FIG. 7D), 2′RA-NRAN01 (closed squares, FIG. 7D), 13′RA-NRAN01 (closedtriangles, FIG. 7D), as described above in regard to FIG. 7B. In thepresent studies, the A375 cells were incubated for 5 minutes in the testagent, washed, and then returned to culture for a 16-18 hour recoveryperiod (i.e., overnight; o/n Recovery), after which time proteinsynthesis was evaluated during a 4 hour ³H-leucine protein synthesisassay. The results of the experiments are plotted in a manner similar tothose described above in regard to FIG. 7A.

The results presented in FIG. 7C and FIG. 7D show the short-term andlong-term effects, respectively, of RA, 2′RA-HS-NR-AN-01 and13′—RA-HS-NR-AN-01 on protein synthesis by A375 target cells. Treatmentof target cells with either the 2′ or 13′RA-NR-AN-01 therapeuticconjugate resulted in a short-term inhibition of protein synthesis,i.e., observed immediately after the 5-minute pulse treatment (FIG. 7C).These findings, when combined with the findings in FIG. 7A and FIG. 7B,above, suggest that the RA-NR-AN-01 conjugates were active and that theywere specifically inhibitory for target cells but not non-target cells.Interestingly, when “pulse” treated target cells were returned toculture no long-term inhibitory effects were observed (FIG. 7D). Theresults presented in FIG. 7C and FIG. 7D again show that Roridin A isnon-specifically inhibitory to test cells (i.e., in a manner similar toFIG. 7A and FIG. 7B, above) and that its effect on the cells is manifesteven after a 16-18 hour recovery period. Thus, the specific effects ofthe RA-NR-AN-01 conjugates on target cells during a “pulse” treatmentappear to be a property of the NR-AN-01 binding protein.

The results obtained with BO54 arterial smooth muscle cells were similarto those obtained with the A375 cells, above, i.e., free Roridin Ashowed a dose-response inhibition of protein synthesis in the short-termequated to be 60%, 66%, and 90% of control at 200 ng/ml, 100 ng/ml, and50 ng/ml; and in long-term the effects on protein synthesis were equatedto be 27%, 46%, and 98% of control at the same dosages. In contrast, the2′ or 13′RA-NR-AN-01 showed only 10-20% inhibition for short- orlong-term effects on protein synthesis (i.e., >80% of control).

Thus, the results show a short-term specific reversible effect ofRoridin A-conjugated NR-AN-01 on target cells when delivered as a“pulse” treatment. However, since only protein synthesis was evaluatedin these experiments, it was possible that cellular metabolic activitymight be affected in the cells as a result of the “pulse” treatment.Therefore, additional studies were conducted in which cellular metabolicactivity was evaluated following “pulse” treatment.

Effects after 5 Minutes of Exposure: Metabolic Activity

MTT assays were conducted at 48 hours following a 5-minute exposure oftarget and non-target cells to RA or RA-NR-AN-01 conjugates. Targetcells in these studies included BO54 and A375, and non-target cellsincluded HT29 cells. Sterile 96 well microtiter plates were seeded with2500 cells/well, wrapped in aluminum foil and incubated in a humidifiedchamber containing 5% CO₂/95% air for 16-18 hours. Serial two-folddilutions of Roridin A (RA), 2′RA-HS-NR-AN-01 and 13′RA-HS-NR-AN-01 wereprepared from 400 ng/ml to 780 pg/ml, and 100 ml aliquots of thedilutions were dispensed into duplicate wells. After 5 minutes exposureto the test samples, the cells were washed to remove the test samples,and fresh medium was added. The cells were allowed 48 hours of recoveryprior to testing: i.e., plates were incubated for 48 hours, and thencellular metabolic activity was determined by adding 20 ml/well of a 5mg/ml MTT solution. The plates were covered and incubated at 37° C. for4 hours and then the reaction was developed as described above (seeEXAMPLE 4, above). The dark blue solubilized formazan reaction productwas developed at room temperature after a 16-18 hour incubation. Thesamples were quantified using an ELISA microtiter plate reader at anabsorbance of 570 nm.

FIG. 8A graphically depicts the results of in vitro studiesinvestigating the effects on BO54 marker-positive smooth muscle cells ofa 5 minute exposure to different concentrations of Roridin A (opensquares, FIG. 8A), 2′RA-NR-AN-01 (NRAN01-2′RA; closed diamonds, FIG.8A), or 13′RA-NR-AN-01 (NRAN01-13′RA; closed squares, FIG. 8A). Theexperiments were conducted in a manner similar to those described abovein regard to FIG. 7B, but metabolic activity was assayed by MTT assay,i.e., rather than protein synthesis as in FIG. 7B, and cells were alsogiven 48 hours to recover (rather than 24 hours, as in FIG. 7B). Theresults of the experiments are plotted in a manner similar to thosedescribed (above) in regard to FIG. 7A.

FIG. 8B graphically depicts the results of in vitro studiesinvestigating the effects on A375 m/m marker-positive cells of a 5minute exposure to different concentrations of Roridin A (open squares,FIG. 8B), 2′RA-NR-AN-01 (NRAN01-2′RA; closed diamonds, FIG. 8B),13′RA-NR-AN-01 (NRAN01-13′RA; closed squares, FIG. 8B). The experimentswere conducted (and the results plotted) in a manner similar to thosedescribed above in regard to FIG. 8A.

FIG. 8C graphically depicts the results of in vitro studiesinvestigating the effects on HT29 marker-negative cells of a 5 minuteexposure to different concentrations of Roridin A (open squares, FIG.8C), 2′RA-NR-AN-01 (NRAN01-2′RA; closed diamonds, FIG. 8C),13′RA-NR-AN-01 (NRAN01-13′RA; closed squares, FIG. 8C). The experimentswere conducted (and the results plotted) in a manner similar to thosedescribed above in regard to FIG. 8A.

The results presented in FIGS. 8A-8C show slight differences between thedifferent RA-NR-AN-01 conjugates at the highest doses, but at the lowerdoses the 2′ and 13′RA-NR-AN-01 did not significantly inhibit targetcell (i.e., BO54 and A375) or non-target cell (i.e., HT29) metabolicactivity over the long-term (i.e., 48 hours). Thus, the results suggestthat the short-term inhibition of target cell protein synthesis (FIGS.7C-7D, above) does not result in long-term metabolic effects on thecells, as measurable in MTT assays. That these assays were able todetect metabolic alterations in cells resulting from a 5 minute exposureis evidenced by the results obtained with free Roridin A. In this case,free Roridin A was non-specifically inhibitory to target and non-targetcell types, even when the cells were exposed to the agent for only 5minutes and then returned to culture for the 48-hour recovery period(FIGS. 8A-8C).

Thus, the findings with free Roridin A suggest that the MTT assay wascapable of detecting metabolic alterations induced during a 5-minuteexposure. Taken together these finding suggest that RA-NR-AN-01conjugates can specifically inhibit target cell activity (i.e., proteinsynthesis) when administered in a “pulse” treatment, and that theseeffects were reversible without significant long-term effects on eitherprotein synthesis or cellular metabolic activity (as measured in an MTTassay). These in vitro properties of the RA-NR-AN-01 conjugates werejudged to be highly useful for inhibition of smooth muscle cell activityin vivo. Therefore, animal model studies were next conducted to evaluatethe effects of these therapeutic conjugates in vivo.

Example 6 Determination of Infusion Conditions in an Animal Model

The therapeutic conjugates of the invention are useful for inhibitingstenosis following vascular trauma or disease. In an illustrativeexample, vascular trauma that is induced during angioplasty is treatedduring the surgical procedure by removing the catheter used to performthe angioplasty, and inserting a balloon infusion catheter into thevessel. The infusion catheter is positioned with the instillation port(or, alternatively, a permeable membrane region) in the traumatized areaof the vessel, and then pressure is applied to introduce the therapeuticconjugate. For example, an infusion catheter with two balloons may beused, and when one balloon is inflated on either side of the trauma sitea fluid space is created that can be filled with a suitable infusionfluid containing the therapeutic conjugate. It has been reportedpreviously that infusion of a horseradish peroxidase (HRP) marker enzymeat a pressure of 300 mm Hg over 45 seconds in dog or human coronaryarteries resulted in penetration of the HRP into the vessel wall (6).However, HRP is a smaller molecule than NR-AN-01 and human and dogcoronary arteries are also considerably smaller than the carotid orfemoral arteries in the present domestic pig model system. Experimentswere therefore conducted to determine, in a domestic pig model system,the infusion conditions suitable for delivery of a therapeutic conjugateto the vascular smooth muscle cells in carotid and femoral arteries.Delivery conditions were monitored by evaluating the penetration of thetherapeutic conjugate into the vascular wall, and specific binding ofthe therapeutic conjugate to the vascular smooth muscle cells in thevessel wall.

Using an infusion catheter, the coronary and femoral arteries ofdomestic pigs or non-human primates were infused with NR-AN-01 for 45seconds to 3 minutes at multiple pressures in the range of about 0.4atmospheres (300 mm Hg) to 3 atmospheres. After infusion, the vesselswere flushed with sterile saline and prepared for immunohistochemistryusing HRP-conjugated goat anti-mouse IgG to detect the NR-AN-01 mouseIgG in the vessel wall. It was determined that full penetration wasachieved of NR-AN-01 into these vessel walls at a pressure of 3atmospheres after 3 minutes.

Immunohistology was also used to determine which animal model systemsexpressed the target antigen for NR-AN-01. Vascular tissue sections fromreadily available experimental animal species were exposed to NR-AN-01,washed, and reacted with HRP-conjugated goat anti-mouse IgG. Onlynon-human primates and swine were found to share the 250 kD NR-AN-01target antigen with man.

To determine whether NR-AN-01 could bind in a specific manner to itstarget antigen in vivo, the coronary and femoral arteries of domesticpigs were infused with therapeutic conjugates using an infusioncatheter, the infusion sites were flushed with sterile saline, thesurgical sites were then closed, and the animals were maintained for anadditional 3-5 days. At the end of this time, the vascular infusionsites were excised and prepared for immunohistology, once again usinggoat anti-mouse IgG to identify NR-AN-01. NR-AN-01 was identified in thevessel wall of swine coronary and femoral arteries 3-5 days aftersurgery, and the NR-AN-01 appeared to be associated only with vascularsmooth muscle cells. These findings suggest that NR-AN-01 is capable ofspecifically binding to its target antigen in vivo.

Example 7 Inhibition of Vascular Smooth Muscle Cells In Vivo

Intimal smooth muscle proliferation that follows ballooncatheter-induced trauma is a good model to evaluate the therapeuticefficacy of conjugates for inhibiting smooth muscle cell activity invivo in response to vascular trauma, including restenosis followingangioplasty. Domestic pigs were used to study the effects of NR-AN-01(i.e., termed vascular smooth muscle binding protein or simply VSMBP inthese studies; and therapeutic conjugates with Roridin A are termedVSMBP-RA). The events which normally follow balloon angioplasty in theporcine artery have been described previously (12). In these studies,dilation of the carotid artery using an oversized balloon (balloon:artery ratio approximately 1.5:1) resulted in complete endothelialdenudation over an area of 1.5-2 cm in length. Although this length oftraumatic injury was selected in an attempt to minimize thrombosis,there was still marked platelet deposition and thrombus formation. Theprocedure also resulted in dissection through the internal elasticlamina into the arterial media and necrosis of medial smooth musclecells. Intimal thickening due to smooth muscle proliferation wasapparent 7 days after injury and reached a mean maximum thickness of 85mm at 14 days. The histological appearance of this neointima is verysimilar to the proliferative neointimal tissue of human restenosis (13).

A single dose test protocol was conducted in domestic pigs withNR-AN-01-Roridin A conjugates. Localized administration of the testconjugates, i.e., through a catheter into a region of traumatized vesselconfined by temporary slip ligatures, was designed to reduce systemictoxicity while providing a high level of exposure for the target smoothmuscle cells. This intra-artery route of administration in animal modelstudies simulates the proposed route in human coronary arteries. Thetest protocol was designed as an initial in vivo screening ofintra-arteriolar, site specific, catheter administered, vascular smoothmuscle binding protein (VSMBP) conjugates. Toxicity of free drug wasalso evaluated, i.e., for pathobiological effects on arteriolar smoothmuscle cells. The therapeutically effective dosage of the RoridinA-NR-AN-01 conjugate was determined by in vitro studies, and the properintra-arteriolar administration pressure was determined by administeringfree MAb and MAb conjugates to animals, as described above in Example 7.

Six domestic crossbred swine (Duroc X), weanling feeder pigs ofapproximately 30 pounds body weight, were used in the experiment. Theanimals were randomly assigned to the following treatment regimen whereeach pig has four different treatments divided between the right andleft carotid and femoral arteries, one of which is a PBS control (Tables1-3, below).

TABLE 1 GROUP NO. TREATMENT GROUP MATERIAL DESCRIPTION 1 CONTROL, VSMBPVSMBP, 200 μg/ml in PBS, pH 6.5 2 CONTROL, PBS PBS, pH 6.5, in injectionsterile water 3 CONTROL, DRUG Roridin A, 2.5 μg/ml in PBS, pH 6.5 4TEST, CONJUGATE VSMBP-RA2′ (200 pg/ml VSMBP & 2.5 μg/ml RA) 5 TEST,CONJUGATE VSMBP-RA13′ (200 μg/ml VSMBP & 3.1 pg/ml RA) 6 TEST, CONJ + RAVSMBP-RA2′ (200 μ/ml VSMBP & 2.5 μg/ml RA) PLUS free Roridin A (2.5μg/ml) 7 TEST, CONJ + RA VSMBP-RA13′ (200 μg/ml VSMBP & 3.1 pg/ml RA)PLUS free Roridin A (2.5 μg/ml)

Surgical Procedure

Test conjugates and control compounds were administered as a singleintra-artery infusion at the site of endothelial denuding and traumainduced by a balloon catheter. Both the carotid and femoral arterieswere abraded over 1 cm to 2 cm of endothelium by intraluminal passage ofa 23 cm, size 3 (femoral) and size 4 (carotid) Uresil Vascu-Flo®silicone occlusion balloon catheter (Uresil Technology Center, Skokie,Ill.), sufficiently distended with saline to generate slight resistance.This technique produced slight distension of the artery. Following thistreatment, proximal and distal slip ligatures, 3-0 silk, were placednear the ends of the abraded region, and a size 8 French, Infant FeedingCatheter (Cutter-Resiflex, Berkeley, Calif.) attached to an InflationPro® (USCI, C. R. Bard, Inc., Billerica, Mass.) pressure syringe wasused to administer the test conjugates and control compounds directly tothe denuded segment at a pressure of three atmospheres for threeminutes. The slip ligatures were removed after the three minute exposureperiod and arterial blood flow was re-established. In these studies,branches of the femoral or carotid arteries were ligated with 00 silksuture as required to attain pressurized infusion in the treated region.The largest distal branch of the femoral artery (the saphenous artery)was incised and used as an entry site for the catheters which were thenpassed into the main femoral artery. Following this catheterizationprocedure in the main femoral artery, the secondary branch was ligated.In these cases, ligation or incision was used to allow entry of thecatheters and the opening was then closed with 3 to 4 sutures of 5-0monosilamen polybutester (Novafil, D & G Monofil Inc., Monati, PR).

Follow-Up Procedures

Following surgery, the pigs were kept in 3×5 foot indoor runs withcement floors during the quarantine and surgical recovery periods. Theywere then transferred to indoor/outdoor pens for the remainder of thefive week healing period prior to collection of tissues for histology.

The animals recovered normally from surgery with no evidence ofhemorrhage or inflammation at the surgical sites. All six animals wereexamined 5 to 6 days after treatment with a doppler stethoscope, and allarteries in each of the animals were patent. Post treatment all animalshad normal appetite, activity and weight gain.

Gross Pathology and Histological Evaluation

Five weeks following the traumatization and treatment of the arteries,the animals were sedated with 0.6 ml Telazol® (tiletamine hydrochloride;A.H. Robins Co., Richmond, Va.) and 0.5 ml xylazine (Lloyd Laboratories,Shenandoah, Iowa) per 30 lb body weight by intramuscular injection,heparinized (i.v. 2 ml sodium heparin, 1000 units/ml), and euthanized byi.v. pentobarbital. Both the right and left carotid and femoral arterieswere removed with normal vessel included both proximal and distal to thetreated segment. The arteries were measured and the location ofligatures and gross abnormalities noted. The arteries were transected at2 mm intervals and arranged in order in cryomolds with O.C.T. (optimumcutting temperature) compound (Tissue Tek®, Miles Laboratories Inc.,Elkhart, Ind.) and frozen in liquid nitrogen. The blocks were sectionedat 5 microns and stained with H&E, Massons Trichrome and MovatsPentachrome for morphological studies. Sections were also used forimmunohistological staining of vascular smooth muscle.

Histological examination of the step sections of the arteries revealedmarked inhibition of intimal smooth muscle proliferation in the regionstraumatized and treated with RA-NR-AN-01 conjugates (Table 2). Thisinhibition was evident even at sub-gross evaluation of the vessels. Theinhibition of intimal smooth muscle cell proliferation was produced withminimal or no histological evidence of smooth muscle cell death in theartery wall. A cross-section of one such traumatized artery is providedin FIGS. 9A and 9B.

TABLE 2 INTIMAL SMOOTH MUSCLE PROLIFERATION IN TRAUMATIZED AND TREATEDPORCINE ARTERIES NO. ARTERIES INTIMAL SMC HYPERTROPHY* TREATMENTEVALUATED ave. (range) Control, MAB 4 3.75 (3-4)  Control, PBS 4 4 (4)Control, RA 2 4 (4) Test, 2′ RA (High pressure) 1 1 (1) (Low pressure) 13 (3) Test, 13′ RA (High pressure) 1 1 (1) (Low pressure) 1 1 (1)*Intimal SMC Hypertrophy: intimal smooth muscle cell hypertrophy scoredon a scale from 1+ (minimal) to 4+ (maximal).

The results presented in FIG. 9A show (at 160× magnification) across-sectional of an untreated artery 5 weeks after angioplasty.Dominant histological features of the artery include displacement of theendothelium (see #1 in FIG. 9A) away from the internal elastic lamina(see #2, FIG. 9A), apparently due to intimal smooth muscle proliferation(see #3, FIG. 9A).

The results presented in FIG. 9B show (at 160× magnification) across-section of a treated artery 5 weeks after angioplasty and infusionof the RA-NR-AN-01 therapeutic conjugate. The vessel in this section wassubjected to greater mechanical stresses than the vessel shown in FIG.9A, with multiple sites where the external elastic membrane was rupturedand associated proliferation of smooth muscle cells in the outer layersof the media was observed (i.e., see #4 in FIG. 9B). Treatment withtherapeutic conjugate inhibited intimal hypertrophy, as evidenced by thelack of displacement of the endothelium (see #1, FIG. 9B) from theinternal elastic lamina (see #2, FIG. 9B). Surprisingly, this inhibitoryeffect on intimal smooth muscle cells was accomplished withoutinhibiting hypertrophy of medial smooth muscle cells in the areas wherethe external elastic membrane was ruptured (see #4, FIG. 9B).

This is a highly fortunate result because wound healing proceeds in thetreated vessel without the adverse consequences of intimal hyperplasiaand stenosis, or necrosis of smooth muscle cells in the media.

In these histological studies, comparisons were also made of theeffectiveness of both the 2′ and the 13′-Roridin A conjugate with thefinding that the 13′ conjugate (i.e., 13′RA-HS-NR-AN-01) appeared to bemore active in inhibiting intimal hyperplasia of smooth muscle cellsthan the 2′ conjugate (i.e., 2′ RA-HS-NR-AN-01). In this study, lowpressure infusion of the 13′ conjugate appeared to inhibit smooth muscleproliferation more effectively than high pressure and the 13′ conjugatealso appeared to be more effective than the 2′ conjugate.

In FIG. 9B, therapeutic conjugate administered at the site followingangioplasty resulted in approximately 95% inhibition of the smoothmuscle hypertrophy that restricted the lumen of the untreated vessel(FIG. 9A). Significantly, the therapeutic conjugate exerted its effectson the smooth muscle cells migrating from the medial smooth musclelayers into the intima, without affecting either endothelium, orproducing any signs of necrosis (i.e., cell death) in the smooth musclecells in the medial layers of the arterial wall. Studies also failed toshow any histological signs of mononuclear infiltration or fibrosis suchas might result from toxic effects on the vessel wall. Also, visiblesigns of healing were observed in the intimal layers of treated vesselsand with re-growth of endothelium observed, i.e., endothelial cellsgrowing over the thin layer of smooth muscle cells in the intima thatlie between the endothelium and internal elastic lamina (i.e., #1 and#2, FIG. 9B). These combined histological observations suggest thehighly desirable features of wound healing, re-growth of endothelium andimproved vascular strength following treatment with a therapeuticconjugate that inhibits smooth muscle hyperplasia in the intimal layersof the vessel.

Example 8 Vascular Smooth Muscle Cell In Vitro DNA and Protein SynthesisInhibition

The ability of various therapeutic agents to inhibit DNA synthesis andprotein synthesis in vascular smooth muscle cells was tested. ³H-leucineand ³H-thymidine uptake and cytotoxicity assays were conducted inaccordance with the following protocols.

5 Minute Exposure; ³H-Leucine Uptake:

Vascular smooth muscle cells at 40,000 cells/ml were seeded in sterile24 well plates at 1 ml/well. The plates were incubated overnight at 37°C., 5% CO₂, 95% air in a humidified atmosphere (saturation). Logdilutions of the therapeutic agent of interest were incubated with thevascular smooth muscle cells for 5 minutes or 24 hours. Samples of thetherapeutic agents were diluted in DMEM:F-12 medium (WhittakerBioproducts, Walkersville, Md.) with 5% fetal bovine serum (FBS, GibcoBRL, Gaithersburg, Md.) and 5% Serum Plus® (JRH Biosciences, Lenexa,Kans.). Following therapeutic agent incubation, the solution wasaspirated, and 1 ml/well of 0.5 microcurie/ml³H-leucine in leucine-freeDMEM (Dulbecco's Modified Eagle's Medium) with 5% Serum Plus® was added.The plates were re-incubated overnight at 37° C., 5% CO₂ in a humidifiedatmosphere. The cells were visually graded using an inverted microscopeusing a scoring scale to determine viability and cell number. The 1 to 3grade is based upon percent of cell viability and number compared tocontrol wells, with 3=100%, 2=70%-100% and 1=0%-70%. A record of thisscoring assisted in determining the immediate cytotoxic effect of thetherapeutic agents. The medium was then aspirated, and the cells werewashed twice with cold 5% TCA. 400 microliters of 0.2M NaOH was addedper well, and the plates were incubated for two hours at roomtemperature on a rotating platform. 200 microliters per well of the cellsolution was transferred into plastic scintillation vials (Bio-RadLaboratories), and 4 milliliters of Bio-Safe® II liquid scintillationfluid (Research Products InterCorp., Mount Prospect, III.) was addedprior to vortexing. Vials were counted on a Beckman LS2800 liquidscintillation counter interfaced with Beckman “Data Capture” softwarefor conversion to a Lotus 1-2-3® file and analysis using Lotus 1-2-3®.

5 Minute Exposure; ³H-Thymidine Uptake:

Vascular smooth muscle cells were incubated in complete medium with 5%FBS (Gibco) overnight at 37° C. in a humidified, 5% CO₂ environment insterile 24 well plates. The medium was aspirated from the wells andserum free medium supplemented with growth factors (DMEM: F-12 basalmedium supplemented with growth factor cocktail, catalog number 11884,which contains insulin (5 micrograms/ml), transferrin (5 micrograms/ml)and sodium selenite (5 nanograms/ml), available from Sigma Chemical, St.Louis, Mo.) was added. Cells were incubated in this medium for 24 hours.For a 5 minute therapeutic agent exposure, log dilutions of thetherapeutic agent were incubated with the cells in complete medium.After 5 minutes and medium aspiration, 1 ml/well of 1.0 microcurie/ml³H-thymidine dispersed in complete medium was added. The 24 hourexposure involved incubation of the cells with 1 ml/well of 1.0microcurie/ml of ³H-thymidine dispersed in complete medium and logdilutions of the therapeutic agent being tested. In both exposuretrials, the cells were then incubated overnight at 37° C. in ahumidified, 5% CO₂ environment. The cells were visually scored forviability and cell number. Cells were washed and prepared for transferinto plastic scintillation vials as described for the ³H-leucineprotocol. Vials were counted on a Beckman LS2800 liquid scintillationcounter interfaced with Beckman “Data Capture” software for conversionto a Lotus 1-2-3® file and analysis using Lotus 1-2-3®.

These protocols are amenable to use with other target cell populations,especially adherent monolayer cell types.

Morphological Cytotoxicity Evaluation-Pulsed Exposure:

Vascular smooth muscle cells were seeded at 4.0×10⁴ cells/ml medium/wellon a commercially prepared four well slide (Nunc, Inc., Naperville,III.). Enough slides were seeded to accommodate two pulsed exposurelengths (5 minutes and 24 hours) and prescribed increment evaluationpoints (24 hours to 128 hours). All slides were run in duplicate toreveal any assay anomalies. The therapeutic agent was diluted in thesame medium used in the ³H-leucine and ³H-thymidine assays. Each fourwell slide was concentration bracketed to one log greater concentration(well “B”), one log lower concentration (well “D”) of the minimaleffective concentration (well “C”), as determined by the ³H-leucine and³H-thymidine assays described above. As a control for normal morphology,one well (well “A”) was left untreated (medium only). Incubation tookplace in a 37° C., 5% CO₂ humidified incubator. After each of the two (5minutes and 24 hours) exposure points, the therapeutic agent medium wasaspirated from each well, including the untreated well. One milliliterof fresh medium was then added to replace the aspirated medium.Re-incubation followed until each of the incremented evaluation pointswere achieved. At those points, the medium was aspirated andsubsequently replaced with 1 ml of 10% neutral buffered formalin for onehour to allow for proper fixation. These fixed slides were stained byhematoxylin (nuclear) and eosin (cytoplasmic) for morphologic evaluationand grading.

Results:

The results of the 24 hour ³H-leucine protein inhibition assay and the24 hour ³H-thymidine DNA synthesis inhibition assay are shown in FIGS.10A-10D for suramin, staurosporin, nitroglycerin and cytochalasin B,respectively. All of the tested compounds showed an availabletherapeutic range (area under the curve of ³H-leucine assay is greaterthan that resulting from the ³H-thymidine assay), indicating usefulnessin the practice of sustained release dosage form embodiments of thepresent invention. More specifically, the compounds inhibited theability of vascular smooth muscle cells to undergo DNA synthesis in thepresence of 5% FBS to a greater extent than they inhibited proteinsynthesis of vascular smooth muscle cells. The protein and DNA synthesisinhibitory effects of suramin, staurosporin, nitroglycerin andcytochalasin B during a 5 minute and 24 hour pulsed exposure are shownin FIGS. 10 A-D, respectively.

Example 9 Specific Binding and Internalization of Targeted Particles byVascular Smooth Muscle Cells

The ability of vascular smooth muscle cells to bind and internalizeparticles coated with binding protein or peptide was demonstrated withmonoclonal antibody (NR-AN-01) coated gold beads both in vitro and invivo. The vascular smooth muscle cell tissue cultures (BO54), an antigenpositive control cell line (A375) and an antigen negative control cellline (HT29) were incubated with 10 nm gold beads, with one group coatedwith NR-AN-01 and a second, uncoated control group. The cells wereexposed to the beads as monolayer and cell suspension cultures, and wereexamined at six time points (i.e., 1 minute, 5 minutes, 15 minutes, 30minutes, 60 minutes and 24 hours) for binding and internalization byelectron microscopy.

Table 3 shows the results of the experimentation, indicating that thebinding to the cell surface is specific. The relative grading systemused throughout Table 3 represents a subjective assessment of particlebinding, wherein 0=none; 1=minimal; 2=mild; 3=moderate; and 4=marked. Ifaggregates of particles settled on the monolayer surface of both thesmooth muscle cells and the control cells, the particles werenonspecifically internalized by macro and micro phagocytosis. When thecells were maintained in a cell suspension, non-specific internalizationwas minimal or absent. Non-specific adherence of gold beads devoid ofNR-AN-01 to surface mucin produced by HT29 cells was observed, resultingin modest non-specific internalization thereof. Vascular smooth musclecell uptake of NR-AN-01 targeted gold beads was highly specific in cellsuspension cultures.

TABLE 3 Primary vessicle micro/macro Cell Cell phagostasis coatedsecondary endoplasmic Time Grid Product Line Surface pinocytosis pitvessicle lysosome golgi reticulum Cell Monolayer  1 min Aa 05(G) A375 20 0 0 0 0 0 Ba 05(G) HT29 0 0 0 0 0 0 0 C 05(G) BO54 2 1 0 0 0 0 0 Da (G) A375 0 0 0 0 0 0 0 Eb  (G) HT29 0 0 0 0 0 0 0 F  (G) BO54 0 0 0 0 00 0  5 min Ac 05(G) A375 4 1 0 0 0 0 0 Bb 05(G) HT29 0 0 0 0 0 0 0 Ca05(G) BO54 3 0 0 0 0 0 0 Dc  (G) A375 0 0 0 0 0 0 0 Ea  (G) HT29 0 0 0 00 0 0 Fa  (G) BO54 0 0 0 0 0 0 0 15 min Aa 05(G) A375 3 1 0 0 0 0 0 Bb05(G) HT29 0 0 0 0 0 0 0 Ca 05(G) BO54 2 1 0 0 0 0 0 Da  (G) A375 0 0 00 0 0 0 Ea  (G) HT29 0 0 0 0 0 0 0 Fa  (G) BO54 0 0 0 0 0 0 0 30 min A05(G) A375 4 3 0 0 0 0 0 B 05(G) HT29 0 0 0 0 0 0 0 C 05(G) BO54 3 2 0 00 0 0 D  (G) A375 0 0 0 0 0 0 0 E  (G) HT29 0 1 0 0 0 0 0 F  (G) BO54 11 0 0 0 0 0 60 Aa 05(G) A375 4 3 2 3 2 0 1 Ba 05(G) HT29 0 0 0 0 0 0 0Cc 05(G) BO54 3 2 0 2 0 0 1 Da  (G) A375 0 1 0 0 0 0 1 Ec  (G) HT29 1 10 1 0 0 0 Fa  (G) BO54 1 2 0 1 0 0 0 24 hrs Ab 05(G) A375 2 1 1 2 4 0 2Ba 05(G) HT29 0 1 1 2 3 0 0 Cc 05(G) BO54 3 3 1 3 4 1 1 Da  (G) A375 0 30 2 3 0 0 Eb  (G) HT29 0 3 0 3 1 0 0 Fb  (G) BO54 0 2 0 2 3 0 0 CellPellets  1 min  1A 05(G) A375 2 0 0 0 0 0 0  7A 05(G) HT29 0 0 0 0 0 0 013A 05(G) BO54 3 0 0 0 0 0 0  1B  (G) A375 0 0 0 0 0 0 0  7B  (G) HT29 00 0 0 0 0 0 13B  (G) BO54 0 0 0 0 0 0 0  5 min  2A 05(G) A375 3 1 0 0 00 0  8A 05(G) HT29 0 0 0 0 0 0 0 14A 05(G) BO54 2 1 0 0 0 0 0  2B  (G)A375 0 0 0 0 0 0 0  8B  (G) HT29 0 0 0 0 0 0 0 15B  (G) BO54 0 0 0 0 0 00 15 min  3A 05(G) A375 4 1 0 1 0 0 0  9A 05(G) HT29 0 0 0 0 0 0 0 15A05(G) BO54 1 1 0 0 0 0 0  3B  (G) A375 0 0 0 0 0 0 0  9B  (G) HT29 0 0 00 0 0 0 15B  (G) BO54 0 0 0 0 0 0 0 30 min  4A 05(G) A375 4 2 0 0 0 0 010A 05(G) HT29 0 0 0 0 0 0 0 16A 05(G) BO54 2 1 0 0 0 0 0  4B  (G) A3750 0 0 0 0 0 0 10B  (G) HT29 0 0 0 0 0 0 0 16G  (G) BO54 0 0 0 0 0 0 0 60 5A 05(G) A375 3 3 0 2 1 0 0 11A 05(G) HT29 0 0 0 0 0 0 0 17A 05(G) BO542 2 0 2 0 0 0  5B  (G) A375 0 0 0 0 0 0 0 11B  (G) HT29 0 0 0 0 0 0 017B  (G) BO54 0 0 0 0 0 0 0 24 hrs  6A 05(G) A375 3 1 0 3 3 0 0 12A05(G) HT29 0 0 0 0 0 0 0 18A 05(G) BO54 2 1 0 1 3 0 0  6B  (G) A375 0 00 0 0 0 0 12B  (G) HT29 1 2 0 2 2 0 0 18B  (G) BO54 6 0 0 0 0 0 0

FIG. 11 shows a tangential section parallel to the inner surface of asmooth muscle cell characterized by numerous endocytic vesicles, severalof which contain antibody coated gold beads in the process of beinginternalized by the cell. These endocytic vesicles with particlesattached to cell surface antigens were stimulated to fuse with lysosomesat a higher than expected rate for normal cell surface membranerecycling. The resultant marked accumulation of internalized particleswas observed at the 24 hour time point and is shown in FIG. 12.

The targeted gold bead vascular smooth muscle cell surface binding,internalization and lysosome concentration was observed in vivo as well.NR-AN-01 coated gold beads were infused via intravascular catheter, openended with treated area occluded proximally and distally with slipligatures, at 3 atm pressure applied for 3 minutes into the wall of apig femoral artery immediately following balloon trauma. The beadinternalization rate varied with the degree of damage sustained by thevascular smooth muscle cell during the balloon trauma. Cells withminimal or no damage rapidly internalized the particles by endocytosisand phagocytosis, concentrating the internalized particles in lysosomes.Cells that were killed by the trauma exhibited surface bead binding.Cells that were damaged by the trauma but survived were characterized bybead surface binding with delayed internalization and lysosomeconcentration. FIG. 13 shows particulate concentration in the lysosomesin vivo at one week following bead administration.

Example 10 Vascular Smooth Muscle in Vitro DNA and Protein SynthesisInhibition by Staurosporin and Cytochalasin

The ability of staurosporin and cytochalasin to inhibit in vitro DNA andprotein synthesis in vascular smooth muscle cells was tested ³H-leucineand ³H-thymidine uptake and cytotoxicity assays were conducted inaccordance with the following protocols.

Cultured Cells:

BO54 cells (baboon smooth muscle cells) were derived from explants ofaortic baboon smooth muscle cells. Cells were expanded in DMEM(Dulbecco's Modified Eagle's Medium):F-12 medium (Mhittaker Bioproducts,Walkersville, Md.) with 5% fetal bovine serum (FBS, Gibco) and 5% SerumPlus® (JRH Biologicals) (“complete medium”), and a seed lot of cells wasfrozen in liquid nitrogen for future use at passage seven.

5 Minute Exposure; Protein Synthesis Assay:

Vascular smooth muscle cells at 40,000-50,000 cells/ml were seeded andprocessed as described in Example 8, “5 minute exposure; ³H-leucineuptake.” Log dilutions of staurosporin (200 ng/ml, 20 ng/ml, 2 ng/ml 0.2ng/ml and 0.02 ng/ml) were dispersed in complete medium. Forcytochalasin B, log dilutions at 20 μg/ml, 2.0 μg/ml, 0.2 μg/ml, 0.02μg/ml and 0.002 μg/ml were dispersed in complete medium. Complete mediumwas then added to the control wells. One ml/well of each therapeuticagent dilution was added in quadruplicate wells, and the agent ofinterest was incubated with the vascular smooth muscle cells for 5 minat room temperature in a sterile ventilated hood. Following therapeuticagent incubation, the wells were subsequently treated as described inExample 8, “5 minute exposure; ³H-leucine uptake.”

5 Minute Exposure; DNA Synthesis Assay:

Vascular smooth muscle (BO54) cells were seeded and processed in 24 wellplates, as described above under “5 Minute Exposure: Protein SynthesisAssay.” After 5 min incubation with the test therapeutic agent, themedium was aspirated and 1 ml/well of 1.0 μCi/ml ³H-thymidine (ratherthan ³H-leucine) dispersed in complete medium was added. The cells werethen incubated overnight at 37° C. in a humidified, 5% CO₂ environment.The toxic effect of the therapeutic agent was then determined, asdescribed in the Protein Synthesis Assay, above.

24 and 120 Hour Exposure; Protein Synthesis Assay:

Vascular smooth muscle (BO54) cells at 20,000 cells/ml were seeded insterile 24 well plates and incubated in complete medium (1 ml/well)overnight at 37° C., 5% CO₂, 95% air in a humidified atmosphere(saturation). Log dilutions of staurosporin (100 ng/ml, 10 ng/ml, 1ng/ml, 0.1 ng/ml and 0.01 ng/ml) were dispersed sequentially in the twomedia, as described below. For cytochalasin B, log dilutions at 10μg/ml, 1.0 μg/ml, 0.1 μg/ml, 0.01 μg/ml and 0.001 μg/ml were dispersedsequentially in the two media, as described below:

Medium (1)=Complete medium; and

Medium (2)=DMEM (leucine-free) with 0.5 μCi/ml ³H-leucine. Medium (2) isused for the final 24 hour incubation period of the experiment.

More specifically, in the 24 hour assay, each therapeutic agent wasdiluted in Medium (2), as noted above. Medium (1) was aspirated from thewells, and aliquots of therapeutic agent dilutions in Medium (2) wereadded in quadruplicate to the appropriate wells. Medium (2) was thenadded to the control wells.

In the 120 hour assay, each therapeutic agent was diluted in Medium (1),as noted above. Medium (1) was aspirated from the wells, and aliquots oftherapeutic agent dilutions in Medium (1) were added in quadruplicate tothe appropriate wells. Medium (1) was then added to the control wells.The medium was changed every 24 hours, and fresh therapeutic agent wasadded to the test wells. At 96 hr, (i.e., the fourth day), eachtherapeutic agent was diluted in Medium (2), as noted above. Medium (1)was aspirated from the wells, and aliquots of therapeutic agentdilutions in Medium (2) were added in quadruplicate to the appropriatewells. Medium (2) was then added to the control wells.

The test agents in ³H-leucine (and controls) were incubated overnight at37° C., 5% CO₂ in a humidified atmosphere. The toxic effect of thetherapeutic agents was then determined, as described in the 5 MinuteExposure: Protein Synthesis Assay, described above. In addition, thechanges in cells at each dilution were photographed using a Zeissmicroscope (Zeiss, West Germany) at 320×. The medium was then aspirated,and the cells were processed with TCA, as described above.

24 and 120 Hour Exposure; DNA Synthesis Assay:

This assay was performed according to the procedure described for “24and 120 Hour Exposure; Protein Synthesis Assay”, except Medium (2) inthis 24 & 120 hr DNA

Synthesis Assay is:

Medium (2)=Complete Medium with 1.0 μCi/ml ³H-thymidine. Medium (2) isused in the final 24 hour incubation of the experiment.

These protein and DNA synthesis assays are amenable for use with othertarget cell populations, especially adherent monolayer cell types.

Results: The minimum effective dose (MED) of each agent was determinedas a percentage of the control that was treated with medium only; 50% ofcontrol values was chosen as the cytotoxicity benchmark. At a 5 minexposure, staurosporin demonstrated an MED of 100 ng/ml in the proteinsynthesis assay and 1 ng/ml in the DNA assay. The 24 hour MED forstaurosporin was 10 ng/ml in the protein synthesis assay and 1 ng/ml inthe DNA synthesis assay. Both assays gave an MED of 1 ng/ml for a 120hour exposure of staurosporin.

At a 5 minute exposure, cytochalasin B demonstrated an MED of 10 μg/mlin the protein synthesis assay as well as in the DNA assay. The 24 hourMED for cytochalasin B was 1.0 μg/ml in the protein synthesis assay and0.1 μg/ml in the DNA synthesis assay. Both assays gave an MED ofapproximately 0.1 μg/ml for a 120 hour exposure of staurosporin.Cytochalasin C and cytochalasin D therapeutic agents were tested at 24and 48 hour exposures using the same dilutions as described forcytochalasin B, above. At 24 hours, cytochalasin C demonstrated an MEDof 1.0 μg/ml in the protein synthesis assay and an MED of 0.01 μg/ml inthe DNA synthesis assay. At 48 hours, cytochalasin C demonstrated an MEDof 0.1 μg/ml in the protein synthesis assay and 0.01 μg/ml in the DNAsynthesis assay. Cytochalasin D demonstrated an MED of 1.0 μg/ml in the24 hour protein synthesis assay and an MED of 0.1 μg/ml in the 24 hr DNAsynthesis assay. A 48 hour exposure to cytochalasin D gave an MEDranging between 0.1 and 0.01 μg/ml in both the protein synthesis and DNAsynthesis assays.

Example 11 Vascular Smooth Muscle Cell Migration Inhibition

Scratch assays to determine the extent of smooth muscle cell migrationinhibition by cytochalasin B were performed in accordance with thefollowing protocol:

Vascular smooth muscle cells (BO54) were derived from explants of baboonaortic smooth muscle, as described in Example 10. The cells were grownin flat bottom, six well tissue culture plates, which hold about 5 ml ofmedium. The vascular smooth muscle cells were plated at 200,000cells/well and placed at 37° C. in a humidified 5% CO₂ incubator for 18hours. The wells were then scratched with a sterile portion of a singleedge razor blade that was held by clamp or pliers and was broughtaseptically into contact with the bottom of the well at a 90° angle. Thecells from a small area along the scratch were removed by a sterilecotton tipped applicator while the blade was in contact with the bottomof the well. After incubation, the presence of cells in the “scratched”area is indicative of cell migration across the scratch line. A controlincubation showed significant cellular migration, and serves as thestandard against which the migration of cells exposed to the therapeuticagent is compared.

Briefly, a stock solution of cytochalasin B (Sigma Chemical Co.) indimethyl sulfoxide (DMSO) at 1 mg/ml was prepared. Test dilutions ofcytochalasin B or control medium were added. Each experiment includedtwo sets of plates:

A set: Test agent exposure for 1, 3, 6, 8 and 10 days only; and

B set: Test agent exposure for 1, 3, 6, 8 and 10 days, followed by aseven day recovery time with control medium.

Both sets of plates were fixed (10% formalin in PBS) and stained (0.02%crystal violet) at the end of the timed exposures. Test concentrationsfor cytochalasin B were 1, 0.1 and 0.01 μg/ml, and a negative mediumcontrol was included. Fresh medium and drug were supplied 3 times perweek.

Table 4 shows the results of these experiments. In this Table, “M”indicates Migration Grade, wherein −=no migration; +1=minimal; +2=mild;+3=moderate; and +4=marked (maximum density; limit of cell contactinhibition) migration of vascular smooth muscle cells into the clearedarea adjacent to the scratch. In this Table, “T” denotes a morphologicalToxicity Grade, wherein −=no toxicity; +1=minimal; +2=mild; +3=moderate;and +4=marked toxicity. The migration results are expressed as “Grade inthe Cleared Area of the Well/Grade in an Undisturbed Region of theWell.” The toxicity values represent a grade for all cells in each well.

The data indicate that cytochalasin B inhibits the migration (+1 to +2)of vascular smooth muscle cells into the cleared area adjacent to thescratch at a dose of 0.1 μg/ml with only minimal (− to +1) morphologicaltoxicity. The data also show that the treated cells (0.1 μg/ml) regainthe ability to migrate (+3 to +4) following removal of the therapeuticagent, even after 10 days of continuous exposure to the therapeuticagent.

TABLE 4 SCRATCH-MIGRATION ASSAY: INHIBITION OF VASCULAR SMOOTH MUSCLECELL MIGRATION BY CYTOCHALASIN B Continuous Exposure 7-day Recovery PostExposure Dosage μg/mL Dosage μg/mL Control Control Day 0.0 0.01 0.1 1.00.0 0.01 0.1 1.0 1 M +1/+3 +1/+3 +1/+3 −/+2 +3/+4 +3/+4 +3/+4 +2/+3 T —— — +3 — — — +2 3 M +3/+4 +3/+4 +1/+4 −/+2 +3/+4 +3/+4 +3/+4 +2/+3 T — —+1 +3 — — — +1 6 M +3/+4 +3/+4 +2/+4 −/+2 +4/+4 +4/+4 +3/+4 +2/+3 T — —+1 +4 — — — +3 8 M +3/+4 +3/+4 +2/+4 −/+2 +4/+4 +4/+4 +3/+4 +2/+3 T — —+1 +4 — — — +3 10 M  +3/+4 +3/+4 +2/+4 −/+2 +4/+4 +4/+4 +4/+4 +2/+3 T —— +1 +4 — — — +3

Example 12 Therapeutic Agent Cytotoxic Effects on Vascular Smooth MuscleCells—Pulse and Continuous Exposure

Vascular smooth muscle cells were exposed to a therapeutic agent in oneof two exposure formats:

Pulse Exposure:

The pulse exposure protocol is described in Example 8 above (see“Morphological Cytotoxicity Evaluation—Pulsed Exposure”).

Continuous Exposure:

The same methodology is used for continuous exposure morphologicalcytotoxicity evaluation as for the pulse exposure, except that the testwells were continuously exposed to therapeutic agent in medium duringthe exposure period. The medium and therapeutic agent were aspiratedfrom each well daily, including from the untreated control well, andwere replaced with 1 ml of fresh medium and therapeutic agent (or mediumalone for control wells). Re-incubation followed, until each of theincremental evaluation points of the long term continuous exposureprotocol was achieved. These incremental evaluation time points were at6, 24, 48, 72, 96, 120, 168, 216 and 264 hours. At the designated timeperiod, the appropriate cells were fixed, stained and evaluated as inthe pulse exposure protocol. The results of a continuous exposureexperiment are shown in Table 5 for suramin, staurosporin andcytochalasin B. The 5 min and 24 hr data presented in Table 5 arecorrelates of the data contained in FIGS. 10A, 10B and 10C.

TABLE 5 MORPHOLOGICAL CYTOTOXICITY ASSAY Drug & Dose Cytochalasin BSuramin Staurosporine Exposure Protocol 10 μg 1 μg 0.1 μg 0.01 μg 10 mg1 mg 0.1 mg 0.01 mg 100 ng 10 ng 1 ng 0.1 ng  5 min + 2 hrs 0.5 0 0 — 00 0 — 0 0 0 —  5 min + 6 hrs 4 1 0 — 1 0 0 — 0 0 0 —  5 min + 24 hrs 40.5 0 — 1 0 0 — 0 0 0 —  5 min + 48 hrs 4 1 0 — 2 0 0 — 2 1 0 —  5 min +72 hrs 4.5 1 0 — 3 1 0 — 3 1.5 0 —  5 min + 96 hrs 5 1 0 — 3 1 0 — 3.51.5 0 —  5 min + 120 hrs 5 1 0 — 1 0 — — 4 1.5 0 — Continuous 6 hrs — 30 0 3 1 0 — 0 0 0 0 Continuous 24 hrs — 3 1 0 3 2 0 — — 0 0 0 24 hrs +24 hrs — 3 0.5 0 4 3 0 — — 0.5 0 0 24 hrs + 48 hrs — 4 1 0 4 3 0 — — 2 00 24 hrs + 72 hrs — 4 0.5 0 4 3 0.5 — — 1 0 0 24 hrs + 96 hrs — 4 0 0 43.5 1 — — 1.5 0 0 24 hrs + 120 hrs — 4 0 0 — — — — — 1.5 0 0 Continuous24 hrs — 3 0 0 — 1 1 0 — 3 1 0 Continuous 48 hrs — 3 1 0 — 3 2 0 — 3 2 0Continuous 72 hrs — 3 1 0 — 4 3 0 — 3 2 0 Continuous 96 hrs — 3 2 0 — 43 0 — 3 2 1 Continuous 120 hrs — 3 1 0 — 5 4 0 — 3 2 1 Continuous 168hrs — 4 1 0 — 5 4 0 — 3 2 1 Continuous 216 hrs — 4 1 0 — 5 4 0 — 3 2 1Continuous 264 hrs — 4 1 0 — 5 4 0 — 4 2 1

At an in vitro effective dosage, cytochalasin B (1 μg/ml; ananti-migration/contraction effective dose) and staurosporin (1 ng/ml; ananti-proliferative effective dose) exhibited a cytotoxicity grade of 1(minimal) and 2 (mild), respectively. Independent studies have indicatedthat a grade of 3 (moderate) or less is preferred for a cytostatic,anti-proliferative agent of the present invention.

Example 13 In Vivo BRDU Assay: Inhibition of Vascular Smooth Muscle CellProliferation

BRDU Assay:

In vivo vascular smooth muscle proliferation was quantitated bymeasuring incorporation of the base analog 5-bromo-2′-deoxyuridine(BRDU, available from Sigma Chemical Co.) into DNA during cellular DNAsynthesis and proliferation. BRDU incorporation was demonstratedhistochemically using commercially available anti-BRDU monoclonalantibodies. The 1 hour pulse labeling permits assessment of the numberof cells undergoing division during the pulse period.

The BRDU pulse labeling protocol described above is used as a standardevaluation technique with in vivo pig vascular studies. Followingsurgical and treatment procedures (discussed, for example, in Examples 7and 11 herein) and a post-surgical recovery period, pigs were sedatedand pulsed with BRDU 1 hour prior to tissue collection.

Briefly, the pigs were sedated with tiletamine hydrochloride andxylazine (as in Example 7, “Gross Pathology and HistologicalEvaluation”) by intramuscular injection. BRDU was then administeredintravenously via the lateral ear vein. Two ml of BRDU at aconcentration of 50 mg/ml was administered to each 30-40 lb pig. Onehour later, the pigs were sacrificed by intravenously administeredpentobarbital. Test artery segments were then removed (a segmentincluded normal vessel located proximally and, if possible, distallywith respect to the treated artery segment). The artery segments weretransected at 2 mm intervals; arranged in order in cryomolds with O.C.T.(optimum cutting temperature) compound (Tissue Tek®, Miles Laboratories,Inc., Elkhart, Ind.); and frozen in liquid nitrogen. The blocks weresectioned at 5 microns and immunohistologically stained to detect BRDUusing the following procedure.

BRDU-labeled cell detection: After BRDU (1 g BRDU diluted in 17 mlsterile water and 3 ml 1 N NaOH) pulse labeling and test artery segmentremoval and sectioning (as above), immunohistochemical staining withanti-BRDU monoclonal antibody provides a visual means of determining amitotic index over a specified time period. The immunohistochemicalstaining method was performed as follows:

1) 5 μg sections of test artery were dehydrated in cold acetone (−20°C.) for 10 minutes;

2) Sections were mounted on glass microscope slides, and the slides weredried in a 37° C. oven for 10 minutes;

3) Slides were rehydrated in PBS for 10 minutes;

4) Slides were subjected to Feulgen's acid hydrolysis using 1 N HCl,wherein two aliquots of 1 N HCl are preheated to 37° C. and 60° C. priorto proceeding;

5) Slides were rinsed with 1 ml of 1 N HCl at 37° C. for 1 min;

6) Slides were transferred to 60° C. 1 NHCL for 15 min;

7) Slides were rinsed with 1 ml of 1 N HCl at 37° C. for 1 min;

8) Slides were washed with room temperature PBS, using 3 changes of PBSat 5 mm intervals;

9) Endogenous, cross-reactive sites on the sections were blocked withnormal goat serum (1:25 in PBS) for 20 min;

10) Slides were washed with PBS, as in step 8;

11) Sections were incubated with mouse anti-BRDU antibody (DAKOCorporation, Carpinteria, Calif.) at 10 μg/ml for 30 min;

12) Slides were washed with PBS, as in step 8;

13) Sections were incubated with horseradish peroxidase-labeled (HRPO)goat anti-mouse IgG, (Jackson Immunoresearch Laboratories, Inc., WestGrove, Pa.; diluted 1:20 in PBS) and 4% human AB serum for 30 min;

14) Slides were washed with PBS, as in step 8;

15) Sections were incubated with chromogen (3,3′-diaminobenzidine (DAB;Sigma) at 5 mg/ml in 200 ml PBS) and 200 μl of 30% H₂O₂ for 10 min;

16) Slides were washed with PBS, as in step 8;

17) Samples were counterstained with Gill I hematoxylin (Gill I LernerLaboratories, Pittsburgh, Pa.; 30 dips);

18) Slides were washed with PBS, as in step 8; rinsed with a bluingsolution (1 gm lithium carbonate in 500 ml dH₂O); washed with deionizedwater; and

19) Test samples were then dehydrated, cleared and coverslipped.

At the conclusion of this procedure, a positive immunohistological stainexhibits a brown color at the site(s) of reactivity.

Cytocidal agents inhibited BRDU uptake relative to a PBS control;however, cytochalasin B and staurosporin inhibited BRDU uptake (i.e.,cell proliferation) without killing the vascular smooth muscle cells.The number of vascular smooth muscle cells labeled with BRDU wasassigned a grade at 400× magnification as follows:

1=≦1/high power field (HPF);

2=2 to 5/HPF;

3=>5 to ≦10/HPF; and

4=>10/HPF.

Both cytochalasin B and staurosporin inhibited proliferation for 24hours following balloon trauma (grade 1), yielding a BRDU labeling gradeequivalent to that of a pre-trauma baseline (grade 1). PBS andmonoclonal antibody controls exhibited grade 2.5 to 4 BRDU labelingduring the same time period. At 4 days post-trauma, arteries treatedwith cytochalasin B or staurosporin, as well as PBS and monoclonalantibody controls, exhibited a BRDU labeling grade of 4. Theanti-proliferative, non-cytocidal properties of cytochalasin B andstaurosporin suggest that these agents are amenable to sustained releasedosage formulations for reduction of vascular stenosis.

Example 14 Biological Stenting of Balloon Traumatized Pig Arteries UsingCytochalasin B

Balloon traumatized pig arteries that had been treated with cytochalasinB displayed a larger luminal area at the 4 day and 3 week post-treatmenttime points, as compared to arteries treated with other test agents orcontrols. Ten femoral arteries (two arteries obtained from each of the 5pigs that were treated according to the single dose protocol describedin Example 7) were evaluated histologically. The maximal luminal area ofeach artery was measured and calculated from digitized microscopicimages by a BQ System IV computerized morphometric analysis system (R &M Biometrics, Inc., Nashville, Term.). This experiment was repeated with5 additional pigs (two arteries per pig; cytochalasin B dose=0.1 μg/ml,applied for 3 min at 1 atm pressure; same time points). The dataobtained from the two experiments were combined. An increase in lumenarea at the 3 week post-cytochalasin B treatment time point wasobserved.

The luminal area of the traumatized and cytochalasin B-treated segmentsof the arteries were also compared to the luminal area of the normal,untreated region of the femoral artery proximal to the test area. Theresults showed that the lumen area in the test region was approximatelytwo times as large as the area of the normal control segment of the sameartery. The negative control agents, PBS and monoclonal antibodyNR-AN-01, showed no increase or a slight decrease in lumen area ascompared to the normal control segment of the same artery.

A cytochalasin B dose response study was then conducted on 10 pigs,following the experimental protocol described in Example 7. Briefly,both arteries in each of 2 pigs were treated with one of the followingdoses of cytochalasin B: 0.0 μg/ml (i.e., PBS negative control); 0.01μg/ml; 0.10 μg/ml; 1.0 μg/ml; and 10.0 μg/ml. The agent was delivered byintraluminal catheter at 1 atm pressure for 3 min, and the arteries wereevaluated 3 weeks later by the morphometric analysis system describedabove. The ratio of treated artery luminal area to proximal normalartery luminal area was determined as a percent change in treated vs.normal area. A significant threshold effect was observed at doses from0.1 μg/ml (≈140% increase) to 1.0 μg/ml (FIG. 14). The 10 μg/ml doseappeared to be toxic to the vascular smooth muscle cells (data notshown). The subthreshold dose (0.01 μg/ml) and negative control (PBS)exhibited a±≈20% change in luminal area. These data suggest thatcytochalasin B acts as a “biological stent” when delivered totraumatized arteries.

Example 15 Direct Conjugation of NR-AN-01 Antibody to CarboxylicFunctional Groups of a Latex Particle

Antibody-coated latex particles (a model of an antibody-coated,sustained release dosage form) may be obtained using the followingaseptic technique:

Conjugation:

To 4 ml 0.05 M sodium borate, pH 8.5, containing 0.01% Tween-20®(polyoxyethylene sorbitan monolaurate, Sigma) is added 0.5 ml PBScontaining 5 mg NR-AN-01 monoclonal antibody. To this solution at roomtemperature is added, with vortexing, 2.5 ml of an aqueous suspensioncontaining 50 mg of 1 μm diameter carboxylated latex particles.Immediately thereafter, 0.50 ml of water containing 100 mg of freshlydissolved 1(3-dimethyl-aminopropyl)-3-ethyl carbodiimide HCl is addedwith vortexing. The solution is then incubated with shaking for 1-2 hrat room temperature. The reaction mixture is then diluted with 50 ml of50 mM phosphate buffer, pH 6.6, containing 0.2% gelatin stabilizer(phosphate/gelatin buffer). The mixture is centrifuged at 40,000×g for 2hr at 4-10° C. The supernatant is decanted, and the pellet isresuspended in 50 ml phosphate/gelatin buffer using low level sonicationfor 10 sec. Centrifugation is repeated, and the pellet is resuspendedtwo times, followed by resuspension in the phosphate/gelatin buffer. Theconjugated particles are then lyophilized using standard protocols andsorbitol excipients.

Characterization:

(a) Sizing: Particle size homogeneity is assessed by laser anisotropyor, for particles larger than 1 μm, by microscopic examination.

(b) Specific Binding Assessment: Specific binding to smooth muscle cellsis determined by histological examination of tissue or cell pelletmicrotome slices after incubation of protein/peptide conjugates withconjugated particles, with or without blocker protein/peptide includedin the incubation mixture. Preferred detection techniques include secondantibody assays (i.e., anti-mouse Ig) or competition assays (i.e.,radioscintigraphic detection in conjunction with radioisotopicallylabeled protein/peptide conjugates).

(c) Assessment of the extent of protein/peptide derivitization: Thisdetermination is performed by coating the latex particles withradioisotopically labeled antibody, followed by detection ofradioactivity associated with the coated particles.

The characterization of antibody-coated particles is described in Table6.

TABLE 6 Characterization of NR-AN-01-Coated Latex Particles ParticleOffering of μg Ab Bound/ Ab Molecules Diameter Ab/Particle 5 mg LatexPer Particle 1.2 μm 40,000 42 3520 1.2 μm 84,000 66 5470 0.4 μm 32,00099 3160 0.4 μm 64,000 140 4550 0.1 μm 932 140 65

The particle aggregation effect of pH during antibody conjugation ispresented in Table 7.

TABLE 7 Effect of pH During Antibody Conjugation - Particle AggregationParticle pH* During Particle Aggregation** Diameter Conjugation +Tween20 ® −Tween 20 ® 1.2 μm 8.5  <5% <2.5%  1.2 μm 7.0 ≈20% ≈10% 1.2 μm 5.5 10% 100% 0.4 μm 8.5 <10%  <5% 0.4 μm 7.0 ≈30% ≈20% 0.4 μm 5.5 100% 100%0.1 μm 8.5 <20% <10% 0.1 μm 7.0 ≈50% ≈40% 0.1 μm 5.5 100% 100% *Using 50mM MES (pH 5.5); phosphate (pH 7.0); or borate (pH 8.5) buffer, asdescribed. **As assessed by microscopic examination, on a scale of0-100%.

These data suggest that proteins or peptides may be directly conjugatedwith sustained release dosage forms of the present invention. Morespecifically, poly-lactic/glycolic acid particulates having terminalcarboxylic acid groups will be conjugated according to the proceduredescribed herein or the alternative procedures described in thespecification hereof.

Example 16 Impact of Tamoxifen on Vascular Smooth Muscle Cells and theRelationship thereof to TGF-Beta Production and Activation

Cell Culture, DNA Synthesis Assay and Cell Counting.

Rat vascular smooth muscle cells were cultured after enzymaticdispersion of the aortic media from 12-17 week old Wistar rats asdescribed in Grainger et al., Biochem. J., 277:145-151, 1991. When thecells reached confluence (after about 6 days) the cells were releasedwith trypsin/EDTA (available from Gibco) and diluted 1:2 in Dulbecco'smodification of Eagle's medium (DMEM; available from ICN/Flow)supplemented with 100 U/ml penicillin and 10% fetal calf serum (FCS).The cells were then replated on tissue culture plastic (available fromICN/Flow) at approximately 1×10⁴ cells/cm². The cells were subculturedrepeatedly in this way when confluence was attained (about every 4days), and the cells were used between passages 6 and 12.

Rat adventitial fibroblasts were cultured as described in Grainger etal., Biochem. J., 283: 403-408, 1992. Briefly, the aortae were treatedwith collagenase (3 mg/ml) for 30 minutes at 37° C. The tunicaadventitia was stripped away from the media. The adventitia wasdispersed for 2 hours in elastase (1 mg/ml) and collagenase (3 mg/ml)dissolved in medium M199 (available from ICN/Flow). The cells were thenspun out (900×g, 3 minutes), resuspended in DMEM+10% FCS and plated outat 8×10⁴ cells/cm² on tissue culture plastic. When the cells reachedconfluence (after about 10 days), they were subcultured as described forvascular smooth muscle cells. Adventitial fibroblasts were subculturedevery 3 days at 1:3 dilution and used between passages 3 and 9.

DNA synthesis was assayed by [³H]-thymidine incorporation as describedin Grainger et al., Biochem. J., 277:145-151, 1991. Vascular smoothmuscle cells were subcultured, grown in DMEM+10% FCS for 24 hours, madequiescent in serum-free DMEM for 48 hours and restimulated with 10% FCSat “0” hours. [³H]-thymidine (5 microcuries/ml; available from AmershamInternational) was added 12 hours after restimulation and the cells wereharvested after 24 hours. DNA synthesis by adventitial fibroblasts wasdetermined similarly, except that the cells were made quiescent inserum-free DMEM for 24 hours.

Cells were prepared for counting by hemocytometer from triplicateculture dishes as described in Grainger et al., Biochem. J.,277:145-151, 1991. Cells were also counted by direct microscopicobservation of gridded culture dishes. The grids were scored into theplastic on the inner surface, so that the cells could not migrate intoor out of the area being counted during the experiment. Cells in each offour squares in two separate wells were counted at each time point. Allcell counting experiments were repeated on at least three separatecultures.

A stock solution of tamoxifen (5 mM; available from ICI Pharmaceuticals)was made up in 10% ethanol (EtOH) and diluted in DMEM and 10% FCS togive the final concentration. The effects of each tamoxifenconcentration were compared with the effects observed in control wellscontaining the same final concentration of the ethanol vehicle.Recombinant TGF-beta (available from Amersham International) wasdissolved in 25 mM Tris/Cl to give a 5 microgram/ml stock solution andsterile filtered through a Spinnex Tube (such as a Centrex DisposableMicrofilter Unit available from Rainin Instrument Company, Inc., Woburn,Mass.). Neutralizing antiserum to TGF-beta (BDA19; available from R & DSystems) was reconstituted in sterile MilliQ water (available fromMillipore Corporation, Bedford, Mass.). At 10 micrograms/ml, thisantibody completely abolished the activity of 10 ng/ml recombinantTGF-beta on subcultured (8th passage) vascular smooth muscle cells.

Assays for TGF-Beta.

The TGF-beta activity present in medium conditioned on various cells wasdetermined by DNA synthesis assay on mink lung endothelial (MvLu) cells;a modification of the assay described in Danielpour et al., J. Cell.Physiol., 138: 79-83, 1989. MvLu cells were subcultured at 1:5 dilutionin DMEM+10% FCS. After 24 hours, the medium was replaced with theconditioned medium to be tested in the absence or presence of theneutralizing antiserum to TGF-beta at 10 micrograms/ml. DNA synthesisduring a 1 hour pulse of [³H]-thymidine (5 microcuries/ml) wasdetermined 23 hours after addition of the test medium. TGF-beta activitywas calculated as the proportion of the inhibition of DNA synthesiswhich was reversed in the presence of neutralizing antibody, using astandard curve to convert the inhibition values into quantities ofTGF-beta. The TGF-betal standards and conditioned media both contained10% FCS in DMEM.

The total latent and active TGF-beta present was determined by asandwich ELISA. Maxisorb 96-well ELISA plates (available from Gibco)were coated with neutralizing antiserum against TGF-beta (BDA19;available from R & D Systems) at 2 micrograms/cm in phosphate bufferedsaline (PBS) overnight at room temperature. The plates were washedbetween each step with tris-buffered saline containing 0.1% Triton X-100(available from Sigma Chemical Company). The plates were incubated withsamples for 2 hours, with a second antibody to TGF-beta (BDA5; availablefrom R & D Systems) at 0.1 micrograms/ml for 2 hours, anti-rabbit IgGperoxidase conjugated to antibody (available from Sigma Chemical Co.),made up according to manufacturer's instructions, for 15 minutes.Absorbances at 492 nm were converted into quantities of TGF-beta proteinusing a standard curve. Both conditioned media and standards wereassayed in the presence of 10% FCS in DMEM. This assay was linear forTGF-beta concentrations in the range from 0.1 ng/ml to 20 ng/ml in thepresence of 10% FCS in DMEM.

RNA Preparation and Northern Analysis. Total cytoplasmic RNA wasisolated from cultured vascular smooth muscle cells as described in Kempet al., Biochem. J., 277: 285-288, 1991. Northern analysis was performedby electrophoresis of total cytoplasmic RNA in 1.5% agarose gels in abuffer containing 2.2 M formaldehyde, 20 mM3-(N-morpholino)propanesulfonic acid, 1 mM EDTA, 5 mM sodium acetate and0.5 micrograms/ml ethidium bromide. The integrity of the RNA was checkedby visualizing the gel under UV illumination prior to transfer ontoHybond N (available from Pharmacia LKB) as specified by themanufacturer. Filters were hybridized as described in Kemp et al.,Biochem. J., 277: 285-288, 1991, using a [³²P] oligolabeled mouseTGF-betal probe corresponding to amino acids 68-228 in the precursorregion of the TGF-betal polypeptide as set forth in Millan et al.,Development, 111: 131-144.

Results.

Vascular smooth muscle cells from the aorta of adult rats proliferatewith a cell cycle time of approximately 35 hours in DMEM+10% FCS (see,for example, Grainger et al., Biochem. J., 277: 145-151, 1991). Additionof tamoxifen decreased the rate of proliferation with maximal inhibitionat concentrations above 33 micromolar. 50 micromolar tamoxifenconcentrations produced an increase in cell number (96 hours followingthe addition of serum) that was reduced by 66%+/−5.2% (n=3). The slowerrate of proliferation was hypothesized to stem from a complete blockageof proliferation for a proportion of the vascular smooth muscle cells orfrom an increase in the cell cycle time of all of the cells. Todistinguish between these possibilities, the proportion of the cellspassing through M phase and the time course of entry into cell divisionwere determined.

Quiescent vascular smooth muscle cells were stimulated with DMEM+10% FCSin the absence or presence of 33 micromolar tamoxifen, with the cellnumber being determined at 8 hour intervals by time lapsephotomicroscopy. In the presence of ethanol vehicle alone, more than 95%of the vascular smooth muscle cells had divided by 40 hours, whereasthere was no significant increase in cell number in the presence oftamoxifen until after 48 hours. By 64 hours, however, more than 90% ofthe cells had divided in the presence of tamoxifen. The time taken for50% of the cells to divide after stimulation by serum was increased from35+/−3 hours (n=7) to 54+/−2 hours (n=3) by 33 micromolar tamoxifen.Since tamoxifen did not significantly reduce the proportion of cellscompleting the cell cycle and dividing, inhibition of vascular smoothmuscle cells caused by tamoxifen appears to be the result of an increasein the cell cycle time of nearly all (>90%) of the proliferating cells.

To determine whether tamoxifen increased the duration of the cell cycleof vascular smooth muscle cells by increasing the duration of the G₀ toS phase, the effect of tamoxifen on entry into DNA synthesis wasanalyzed. Tamoxifen at concentrations up to 50 micromolar did notsignificantly affect the time course or the proportion of cells enteringDNA synthesis following serum stimulation of quiescent vascular smoothmuscle cells (DNA synthesis between 12 hours and 24 hours afterstimulation was measured by [³H]-thymidine incorporation: control at17614+/−1714 cpm; 10 micromolar tamoxifen at 16898+/−3417 cpm; and 50micromolar tamoxifen at 18002+/−4167 cpm). Since the duration of S phaseis approximately 12 hours (unpublished data), tamoxifen does not appearto have significantly impacted the time course of entry into DNAsynthesis. These results therefore imply that tamoxifen decreases therate of proliferation of serum-stimulated vascular smooth muscle cellsby increasing the time taken to traverse the G₂ to M phase of the cellcycle.

Based upon these results, it appeared that tamoxifen exhibited effectssimilar to those previously described for TGF-beta (see, for example,Assoian et al., J. Cell. Biol., 109: 441-448, 1986) with respect toproliferation of subcultured vascular smooth muscle cells in thepresence of serum. Tamoxifen is known to induce TGF-beta activity incultures of breast carcinoma cell lines as described, for example, inKnabbe, et al., Cell, 48: 417-425, 1987. Consequently, experimentationwas conducted to determine whether tamoxifen decreased the rate ofproliferation of vascular smooth muscle cells by inducing TGF-betaactivity. When quiescent vascular smooth muscle cells were stimulatedwith 10% FCS in the presence of 50 micromolar tamoxifen and 10micrograms/ml neutralizing antiserum against TGF-beta, the cellsproliferated at the same rate as control cells in the presence ofethanol vehicle alone.

To confirm that the vascular smooth muscle cells produced TGF-beta inresponse to tamoxifen, such cells were treated with tamoxifen for 96hours in the presence of 10% FCS. The conditioned medium was thencollected and TGF-beta activity was determined by the modified mink lungepithelial (MvLu) cell assay described above. Tamoxifen increased theTGF-beta activity in the medium by >50-fold. Addition of tamoxifen (50micromolar) in fresh DMEM+10% FCS to the MvLu cells had no effect on DNAsynthesis, demonstrating that tamoxifen did not induce production ofactive TGF-beta by the MvLu cells.

TGF-beta is produced as a latent propeptide which can be activatedoutside the cell by proteases such as plasmin. To determine whethertamoxifen increased TGF-beta activity by promoting the activation oflatent TGF-beta or by stimulating the production of the latentpropeptide which was subsequently activated, the total latent plusactive TGF-beta present in the conditioned medium was determined bysandwich ELISA as described above. After 96 hours in the presence oftamoxifen (50 micromolar), the total TGF-beta protein present wasincreased by approximately 4-fold. Furthermore, the proportion of theTGF-beta present in active form was increased from <5% in the mediumconditioned on vascular smooth muscle cells in the presence of ethanolvehicle alone to approximately 35% in the medium conditioned on cellstreated with tamoxifen. Thus, tamoxifen appears to increase TGF-betaactivity in cultures of rat vascular smooth muscle cells by stimulatingthe production of latent TGF-beta and increasing the proportion of thetotal TGF-beta which has been activated.

Heparin increases TGF-beta activity in medium conditioned on vascularsmooth muscle cells (unpublished data). The mechanism of action ofheparin in this regard appears to involve the release of TGF-beta frominactive complexes present in serum, because pretreatment of serum withheparin immobilized on agarose beads is as effective as direct additionof free heparin to the cells. To determine whether tamoxifen acts torelease TGF-beta from sequestered complexes in serum which are notimmunoreactive in the ELISA assay, 10% FCS+DMEM was treated with 50micromolar tamoxifen for 96 hours at 37° C. in the absence of cells.Medium treated in this way contained similar levels of TGF-beta proteinand activity to untreated medium. It appears, therefore, that tamoxifen,unlike heparin, does not act by releasing TGF-beta from inactivecomplexes present in serum.

The content of TGF-betal mRNA was also analyzed by Northern analysis atvarious time points after addition of tamoxifen. Subcultured ratvascular smooth muscle cells (6th passage in exponential growth) in theabsence or presence of ethanol vehicle alone contain very little mRNAfor TGF-betal. By 24 hours after addition of tamoxifen (10 micromolar),TGF-betal mRNA was increased approximately 10-fold.

Although TGF-beta decreases the rate of proliferation of vascular smoothmuscle cells, it does not affect the rate of proliferation offibroblasts. Tamoxifen at concentrations of up to 50 micromolar did notreduce the rate of proliferation of subcultured adventitial fibroblasts.Tamoxifen is therefore a selective inhibitor of vascular smooth muscleproliferation with an ED₅₀ at least 10-fold lower for vascular smoothmuscle cells than for adventitial fibroblasts.

CITATIONS

-   1. Popma, J. J. et al. 1990. Factors influencing restenosis after    coronary angioplasty. Amer. J. Med. 88:16 N-24N.-   2. Fanelli, C. et al. 1990. Restenosis following coronary    angioplasty. Amer. Heart Jour. 119: 357-368.-   3. Johnson, D. E. et al. 1988. Coronary atherectomy: Light    microscopic and immunochemical study of excised tissue (abstract).    Circulation 78 (Suppl. II): II-82.-   4. Liu, M. W. et al. 1989. Restenosis after coronary angioplasty;    Potential biologic determinants and role of intimal hyperplasia.    Circulation 79: 1374-1387.-   5. Clowes, A. W. et al. 1985. Significance of quiescent smooth    muscle migration in the injured rat carotic artery. Circ. Res. 56:    139-145.-   6. Goldman, B. et al. 1987. Influence of pressure on permeability of    normal and diseased muscular arteries to horseradish peroxidase; A    new catheter approach. Atherosclerosis 65: 215-225.-   7. Wolinsky, H. et al. 1990. Use of a perforated balloon catheter to    deliver concentrated heparin into the wall of the normal canine    artery. JACC 15 (2): 475-481.-   8. Nabel, E. G. et al. 1989. Recombinant gene expression in vivo    within endothelial cells of the arterial wall. Science 244:    1342-1344.-   9. Middlebrook, J. L. et al. 1989. Binding of T-2 toxin to    eukaryotic cell ribosomes. Biochem. Pharm. 38 (18): 3101-3110.-   10. Barbacid, M. et al. 1974. Binding of [acetyl-¹⁴C] trichodermin    to the peptidyl transferase center of eukaryotic ribosomes. Eur. J.    Biochem. 44: 437-444.-   11. Sclingemann et al. 1990. Am. J. Pathol. 136: 1393-1405.-   12. Steele P. M., Chesebro J. H., Stanson A. W., et al. 1985.    Balloon angioplasty: natural history of the pathophysiological    response to injury in a pig model. Circ. Res. 57:105-112.-   13. Schwartz, R. S., Murphy J. G., Edwards W. D., Camrud A. R.,    Vliestra R. E., Holmes D. R. Restenosis after balloon angioplasty. A    practical proliferative model in porcine coronary arteries.    Circulation 1990; 82:2190-2200.-   14. Bumol, T. F. and R. A. Reisfeld. 1982. Unique    glycoprotein-proteoglycan complex defined by monoclonal antibody on    human melanoma cells. Proc. Natl. Acad. Sci. USA 79:1245-1249.

While the preferred embodiment of the invention has been illustrated anddescribed, it will be appreciated that various changes can be madetherein without departing from the spirit and scope of the invention.

1-20. (canceled)
 21. A method for inhibiting or treating stenosis orrestenosis comprising administering to a blood vessel of a mammal anamount of taxol or a structural analog thereof via a catheter, whereinthe taxol or structural analog thereof is non-targeted and non-bindingpartner associated.
 22. The method of claim 21, wherein theadministration is local.
 23. The method of claim 21, wherein the bloodvessel is subjected to procedural vascular trauma.
 24. The method ofclaim 23, wherein the administration is before, during or after thevascular trauma.
 25. The method of claim 23, wherein the vascular traumais associated with angioplasty, placement of a stent, or grafting. 26.The method of claim 21 wherein the amount is a cytostatic amount. 27.The method of claim 21 wherein the amount of taxol or a structuralanalog thereof exerts minimal protein synthesis inhibition andcytotoxicity on vascular smooth muscle cells, and exerts significant DNAsynthesis inhibition on the vascular smooth muscle cells.
 28. The methodof claim 21 wherein the amount is effective to inhibit proliferation ofvascular smooth muscle cells.
 29. The method of claim 21 wherein theamount of taxol or structural analog thereof is in a sustained releasedosage form.
 30. The method of claim 29 wherein the sustained releasedosage form comprises microparticles or nanoparticles.
 31. The method ofclaim 30 wherein the microparticles or nanoparticles are in a liquidvehicle.
 32. The method of claim 29 wherein the sustained release dosageform is biodegradable.
 33. A system comprising an amount of taxol or astructural analog thereof, wherein the taxol or structural analogthereof is non-targeted and non-binding partner associated, and acatheter adapted for administering the amount of taxol or structuralanalog thereof to a blood vessel of a mammal, wherein the amount oftaxol or structural analog thereof is effective to inhibit or treatstenosis or restenosis.
 34. The system of claim 33 wherein the amount isa cytostatic amount.
 35. The system of claim 33 wherein the amount oftaxol or a structural analog thereof exerts minimal protein synthesisinhibition and cytotoxicity on vascular smooth muscle cells, and exertssignificant DNA synthesis inhibition on the vascular smooth musclecells.
 36. The system of claim 33 wherein the amount is effective toinhibit proliferation of vascular smooth muscle cells.
 37. The system ofclaim 33 wherein the amount of taxol or structural analog thereof is ina sustained release dosage form.
 38. The system of claim 37 wherein thesustained release dosage form comprises microparticles or nanoparticles.39. The system of claim 38 wherein the microparticles or nanoparticlesare in a liquid vehicle.
 40. The system of claim 37 wherein thesustained release dosage form is biodegradable.